JP6391564B2 - Molecules, compositions and their use with reduced effector function and extended half-life - Google Patents

Description

  The present disclosure provides for molecules comprising variant IgG Fc comprising mutations that result in reduced effector function and extended half-life while maintaining desirable stability, particularly but not limited to immunoglobulins (eg, , Antibodies) and the like. The present disclosure also relates to methods of making and using such polypeptides, expression vectors, host vectors, nucleic acids encoding host cells, and therapeutic and diagnostic compositions, formulations and kits.

  Antibodies are composed of two distinct regions called variable (Fv) regions and constant (Fc) regions. The Fc region of an antibody interacts with several ligands, such as the Fc receptor and C1q, conferring a set of functional capabilities called effector functions. Fc receptors mediate communication between the antibody and the cellular arm of the immune system (Ragavan et al., Annu. Rev. Cell. Dev. Biol. 12: 181-220 (1996); et al., Annu. Rev. Immunol. 19: 275-290, (2001)).

  The formation of Fc / FcγR complexes typically results in signaling events within these cells, as well as subsequent immune responses such as release of inflammatory mediators, B cell activation, endocytosis, phagocytosis, and cells Causes a damaging attack. The cell-mediated reaction that causes lysis of target cells after non-specific cytotoxic cells expressing FcγRs recognize the antibody bound to the target cell is called antibody-dependent cellular cytotoxicity (ADCC) (Ghetie et al. Rev. Immunol. 18: 739-766 (2000); Ravetech et al., Annu. Rev. Immunol. 19: 275-290 (2001)).

  Human FcγRs are divided into three different classes: FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). IgG molecules exhibit differential isotype specificity for FcγR. IgG3 molecules bind strongly to all FcγR isoforms. IgG1, the most common isoform in blood, binds to all FcγRs with low affinity for FcγRIIA / β isoforms. IgG4 is a moderate binding substance for FcγRI and a weak binding substance for FcγRIIB. Finally, IgG2 binds only weakly to one allelic form of FcγRIIA, FcγRIIA-H131 (Siberil et al., J. Immunol. Lett. 106: 111-118 (2006)). A short contiguous segment of amino acid residues (234-238) in the N-terminal part of the CH2 region that are directly involved in the binding of residues 268, 297, 327 and 329 also bind to a subset of FcγR A number of residues located in the CH2 and CH3 regions also contribute to FcγR binding (Canfield et al., J. Exp. Med. 173: 1483-91 (1991), Chappel). et al., Proc. Natl. Acad. Sci. USA 888: 9. 36-40 (1991), Gergely et al.FASEB J.4: 3275-83 (1990)).

  The overlapping site of the Fc region of the molecule controls the activation of complement-mediated cell-independent cytotoxic functions. Thus, Fc binding to complement protein C1q mediates a process called complement-dependent cytotoxicity (CDC) (Ward et al., Ther. Immunol. 2: 77-94 (1995)).

  In some cases it is advantageous to reduce or eliminate effector function. In such cases, it is beneficial to use antibodies or Fc domain-containing fragments that recruit few complement or effector cells (see, eg, Wu et al., Cell Immunol. 200: 16-26). 2000); Shields et al., J. Biol.Chem.276: 6591-6604 (2001); US Pat. No. 6,194,551; US Pat. No. 5,885,573; Publication No. 04/029207; and US 2011/0059078).

  Certain subclasses of human immunoglobulins recruit only a small amount of complement or effector cells, eg, IgG2 and IgG4, but natural immunoglobulins lacking all effector functions are not known. Thus, another approach is to alter or mutate residues in the Fc region responsible for effector function. For example, see the following documents: PCT publication: WO20060776594 pamphlet; WO199958572 pamphlet; WO20066043503 pamphlet, and WO200660553301 pamphlet; US 2006-0134709 U.S. Pat. No. 5,624,821; U.S. Pat. Nos. 6,194,551; and 5,885,573; Armour et al. , Eur. J. et al. Immunol. 29: 2613-2624 (1999); Reddy et al. , J .; Immunol. 164: 1925-1933 (2000); Xu et al. Cell Immunol. 200: 16-26 (2000); Shields et al. , J .; Biol. Chem. 276: 6591-6604 (2001).

  A consideration for the reduction or elimination of effector function is that other important antibody properties are not perturbed. Thus, modification of Fc variants maintains FcγR and the ability to interact with other important Fc ligands such as FcRn and proteins A and G, as well as antibody stability, solubility, and structural integrity. It should be carried out so as to remove only the binding to C1q.

US Pat. No. 6,194,551 US Pat. No. 5,885,573 International Publication No. 04/029207 Pamphlet US Patent 2011/0059078 International Publication No. 2006076594 Pamphlet International Publication No. 199958572 Pamphlet International Publication No. 2006047350 pamphlet, International Publication No. 20060553301 Pamphlet US 2006-0134709 US Pat. No. 5,624,821 US Pat. No. 6,194,551 US Pat. No. 5,885,573

Raghavan et al. , Annu. Rev. Cell. Dev. Biol. 12: 181-220 (1996) Ravetch et al. , Annu. Rev. Immunol. 19: 275-290, (2001)) Ghetie et al. , Annu. Rev. Immunol. 18: 739-766 (2000); Siberil et al. , J .; Immunol. Lett. 106: 111-118 (2006) Canfield et al. , J .; Exp. Med. 173: 1483-91 (1991), Chappel et al. , Proc. Natl. Acad. Sci. USA 888: 9036-40 (1991), Gergely et al. FASEB J.H. 4: 3275-83 (1990)) Ward et al. Ther. Immunol. 2: 77-94 (1995)). Wu et al. Cell Immunol. 200: 16-26 (2000) Shields et al. , J .; Biol. Chem. 276: 6591-6604 (2001) Armour et al. , Eur. J. et al. Immunol. 29: 2613-2624 (1999) Reddy et al. , J .; Immunol. 164: 1925-1933 (2000) Xu et al. Cell Immunol. 200: 16-26 (2000)

  The present disclosure provides recombinant polys comprising a variant Fc domain having amino acid substitutions that result in desired properties such as reduced effector function and improved plasma half-life while maintaining stability, e.g., thermal stability. Relates to peptides. In certain embodiments, a polypeptide of the present disclosure comprises a variant IgG Fc domain, wherein the variant IgG Fc domain is (a) a phenylalanine (F) amino acid at position 234; (b) an alanine (A) at position 235; Asparagine (N), phenylalanine (F), glutamine (Q), or valine (V) amino acid; (c) alanine (A), aspartic acid (D), glutamic acid (E), histidine (H), asparagine at position 322 (N), or glutamine (Q) amino acid; or contains (has) an alanine (A) or glycine (G) amino acid at position 331, where the amino acid numbering is according to the EU index described in Kabat.

  In another embodiment, the polypeptide comprises a phenylalanine (F) amino acid at position 234; a glutamine (Q) amino acid at position 235; and a glutamine (Q) amino acid at position 322, wherein the amino acid numbering is set forth in Kabat. According to the EU index. In some embodiments, the polypeptide comprises a phenylalanine (F) amino acid at position 234; a glutamine (Q) amino acid at position 235; and a glycine (G) amino acid at position 331, wherein the amino acid numbering is as described in Kabat. Follow the EU index. In some embodiments, the polypeptide comprises a phenylalanine (F) amino acid at position 234; an alanine (A) amino acid at position 235; and a glutamine (Q) amino acid at position 322, wherein the amino acid numbering is as described in Kabat. Follow the EU index.

  In some embodiments, the polypeptide further comprises (a) a tyrosine (Y) amino acid at position 252 or a serine (S) amino acid at position 252 or a tryptophan (W) amino acid at position 252 or a threonine (T) amino acid at position 252. And / or (b) a threonine (T) amino acid at position 254; and / or (c) a glutamic acid (E) amino acid at position 256, or a serine (S) amino acid at position 256, or an arginine (R) amino acid at position 256; Or a glutamine (Q) amino acid at position 256, or an aspartic acid (D) amino acid at position 256, where the amino acid numbering is according to the EU index described in Kabat.

  In another embodiment, the polypeptide further comprises (a) a tyrosine (Y) amino acid at position 252; and / or (b) a threonine (T) amino acid at position 254; and / or (c) glutamic acid at position 256 (E). Including amino acids, where the amino acid numbering is according to the EU index described in Kabat. In another embodiment, the polypeptide comprises (a) a tyrosine (Y) amino acid at position 252 or a serine (S) amino acid at position 252 or a tryptophan (W) amino acid at position 252 or a threonine (T) amino acid at position 252. And (b) contains a threonine (T) amino acid at position 254, where the amino acid numbering is according to the EU index set forth in Kabat. In some embodiments, the polypeptide comprises (a) a threonine (T) amino acid at position 254; and (b) a glutamic acid (E) amino acid at position 256, or a serine (S) amino acid at position 256, or an arginine (R) at position 256. ) Amino acids, or glutamine (Q) amino acids at position 256, or aspartic acid (D) amino acids at position 256, where the amino acid numbering is according to the EU index as described in Kabat.

  In some embodiments, the polypeptide further comprises (a) a tyrosine (Y) amino acid at position 252; a serine (S) amino acid at position 252; a tryptophan (W) amino acid at position 252; or a threonine (T) amino acid at position 252; And (b) glutamic acid (E) amino acid at position 256, serine (S) amino acid at position 256, arginine (R) amino acid at position 256, glutamine (Q) amino acid at position 256, or aspartic acid at position 256 ( D) Contains amino acids, where the amino acid numbering is according to the EU index described in Kabat.

  In some embodiments, the polypeptide further comprises (a) a tyrosine (Y) amino acid at position 252 and a threonine (T) amino acid at position 254; or (b) a threonine (T) amino acid at position 254 and a glutamic acid at position 256 (E ) Amino acids; (c) contains a tyrosine (Y) amino acid at position 252 and a glutamic acid (E) amino acid at position 256, where the amino acid numbering is according to the EU index set forth in Kabat. In some embodiments, the polypeptide comprises a tyrosine (Y) amino acid at position 252, a threonine (T) amino acid at position 254, and a glutamic acid (E) amino acid at position 256, wherein the amino acid numbering is as described in Kabat. Follow the EU index.

  In one embodiment, the polypeptide comprises (a) a phenylalanine (F) amino acid at position 234; (b) a glutamine (Q) amino acid at position 235; (c) a glutamine (Q) amino acid at position 322; (d) at position 252. Tyrosine (Y) amino acid; (e) threonine (T) amino acid at position 254; and (f) glutamic acid (E) amino acid at position 256, where the amino acid numbering is according to the EU index set forth in Kabat. In one embodiment, the polypeptide comprises (a) a phenylalanine (F) amino acid at position 234; (b) a glutamine (Q) amino acid at position 235; (c) a glycine (Q) amino acid at position 331; (d) at position 252. Tyrosine (Y) amino acid; (e) threonine (T) amino acid at position 254; and (f) glutamic acid (E) amino acid at position 256, where the amino acid numbering is according to the EU index set forth in Kabat. In another embodiment, the polypeptide comprises (a) a phenylalanine (F) amino acid at position 234; (b) an alanine (A) amino acid at position 235; (c) a glutamine (Q) amino acid at position 322; (d) position 252. Tyrosine (Y) amino acid; (e) threonine (T) amino acid at position 254; and (f) glutamic acid (E) amino acid at position 256, where the amino acid numbering is according to the EU index set forth in Kabat.

  In certain embodiments, the polypeptide has improved pharmacokinetic (PK) properties compared to the same polypeptide comprising a wild type Fc domain. In some embodiments, the PK property is half-life. In certain embodiments, the polypeptide has improved FcRn binding compared to the same polypeptide comprising a wild type Fc domain. In some embodiments, the IgG Fc domain is non-human. In another aspect, the IgG Fc domain is human. In a specific embodiment, the non-human IgG Fc domain is derived from a rodent, donkey, sheep, rabbit, goat, guinea pig, camel, horse or chicken.

In some embodiments, the IgG Fc domain is a human immunoglobulin G class 1 (IgG ₁ ) Fc domain, a human immunoglobulin G class 2 (IgG ₂ ) Fc domain, a human immunoglobulin G class 3 (IgG ₃ ) Fc domain, and a human Selected from the group consisting of immunoglobulin G class 4 (IgG ₄ ) Fc domains. In certain embodiments, the polypeptide further comprises an antigen binding domain. In certain embodiments, the antigen binding domain is derived from a monoclonal antibody or antigen binding fragment thereof. In some embodiments, the antigen binding domain is derived from a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the antigen binding domain comprises (a) a single chain antibody; (b) a diabody; (c) a polypeptide chain of the antibody; (d) an F (ab ′) ₂ fragment; or (e) F (ab ) Containing fragments.

  In certain embodiments, the polypeptide has reduced Fc-mediated effector function compared to the same polypeptide comprising a wild type Fc domain. In certain embodiments, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC). In other aspects, the effector function is complement dependent cytotoxicity (CDC). In some embodiments, the polypeptide has a lower affinity for the Fcγ receptor (FcγR) compared to the same polypeptide comprising a wild type Fc domain. In some embodiments, the FcγR is human FcγR. In some embodiments, the FcγR is selected from the group consisting of FcγRI, FcγRII, and FcγRIII. In some embodiments, the FcγRI is FcγRIα. In another aspect, the FcγRII is FcγRIIa or FcγRIIb. In yet another aspect, the FcγRIII is FcγRIII (158V) or FcγRIII (158F).

  In certain embodiments, the polypeptide binds FcRn with improved affinity compared to the same polypeptide comprising a wild type Fc domain. In some embodiments, the polypeptide has a higher affinity for FcRn at pH 6.0 than at pH 7.4. In certain embodiments, the polypeptide binds C1q with reduced affinity compared to the same polypeptide comprising a wild type Fc domain.

  In certain embodiments, the polypeptide exhibits increased thermal stability compared to the same polypeptide comprising a FES-YTE IgG Fc domain. In some embodiments, the increase in thermal stability is measured by differential scanning calorimetry (DSC). In certain embodiments, the increase in thermal stability is at least 4 ° C. In some embodiments, thermal stability is measured by differential scanning fluorimetry (DSF). In a specific aspect, the DSF fluorescent probe is Sypro Orange. In some embodiments, the increase in thermal stability is at least 5 ° C.

  In certain embodiments, the polypeptide exhibits an increase in apparent solubility as measured using a polyethylene glycol (PEG) precipitation assay as compared to the same polypeptide comprising a FES-YTE IgG Fc domain. In certain embodiments, the polypeptide exhibits an increase in stability as measured using an accelerated stability assay compared to the same polypeptide comprising a FES-YTE IgG Fc domain. In some embodiments, the accelerated stability assay comprises (i) incubation of the polypeptide for an extended period of time, and (ii) incubation at an elevated temperature. In certain embodiments, the accelerated stability assay comprises incubation at a high concentration. In some embodiments, the long period is at least one month. In certain embodiments, the high concentration is at least 25 mg / ml. In certain embodiments, the elevated temperature is at least 40 ° C. In certain embodiments, the accelerated stability assay is performed using high performance size exclusion chromatography (HPSEC) or dynamic light scattering (DLS).

  The present disclosure also provides an isolated nucleic acid comprising a sequence encoding a polypeptide of the present disclosure. Also provided are compositions, expression vectors, and host cells containing a nucleic acid comprising a sequence encoding a polypeptide of the present disclosure. A host cell can contain an isolated nucleic acid comprising a sequence encoding a polypeptide of the present disclosure, a composition containing a nucleic acid comprising a sequence encoding a polypeptide of the present disclosure, or a sequence encoding a polypeptide of the present disclosure. An expression vector containing the nucleic acid to be included can be included.

  The present disclosure produces a polypeptide of the present disclosure comprising: (a) culturing a host cell containing a nucleic acid comprising a sequence encoding a polypeptide of the present disclosure; and (b) isolating the polypeptide. It also provides a way to do this. The present disclosure also provides a composition comprising a polypeptide of the present disclosure and a carrier.

  The present disclosure also provides a diagnostic agent comprising a polypeptide of the present disclosure. In some embodiments, the polypeptide is labeled. The present disclosure also provides conjugates comprising a polypeptide of the present disclosure and a therapeutic moiety. The present disclosure also includes (a) a polypeptide of the present disclosure, (b) an isolated nucleic acid comprising a sequence encoding the polypeptide of the present disclosure, (c) a nucleic acid having a sequence encoding the polypeptide of the present disclosure. (D) a composition comprising the polypeptide of the present disclosure and a carrier; (e) in certain embodiments, a diagnostic agent comprising a polypeptide of the present disclosure that can be labeled; Or (f) kits comprising a conjugate comprising a polypeptide of the present disclosure and a therapeutic moiety are also provided.

  The present disclosure also provides a method of treating a mammal comprising an effective amount of (a) a polypeptide of the present disclosure, (b) a sequence encoding the polypeptide of the present disclosure, and an isolated nucleic acid (C) a composition comprising a nucleic acid having a sequence encoding a polypeptide of the present disclosure, an expression vector or a host cell, (d) a composition comprising a polypeptide of the present disclosure and a carrier, (e) A diagnostic agent comprising a polypeptide of the present disclosure that can be labeled, or (f) a conjugate comprising a polypeptide of the present disclosure and a therapeutic moiety is administered to a mammal in need of treatment.

  The disclosure also provides a method of reducing Fc-induced effector function of a parent polypeptide comprising an Fc domain, the method comprising: (a) replacing the amino acid at position 234 in the Fc domain with phenylalanine (F); b) replacing the amino acid at position 235 in the Fc domain with alanine (A), asparagine (N), phenylalanine (F), glutamine (Q), or valine (V); (c) the amino acid at position 322 of the Fc domain; Alanine (A), aspartic acid (D), glutamic acid (E), histidine (H), asparagine (N), or glutamine (Q); or the amino acid at position 331 of the Fc domain is alanine (A) Or comprising substituting with glycine (G), wherein the amino acid numbering is According to the EU index as set forth in abat. In one embodiment of the above method, the Fc domain of the parent polypeptide comprises (a) a tyrosine (Y) amino acid at position 252 or a serine (S) amino acid at position 252 or a tryptophan (W) amino acid at position 252 or a position 252 Threonine (T) amino acid; and / or (b) threonine (T) amino acid at position 254; and / or (c) glutamic acid (E) amino acid at position 256, or serine (S) amino acid at position 256, or position 256 Contains an arginine (R) amino acid, or a glutamine (Q) amino acid at position 256, or an aspartic acid (D) amino acid at position 256, where the amino acid numbering is according to the EU index set forth in Kabat. In another embodiment of the above method, the Fc domain of the parent polypeptide comprises (a) tyrosine (Y) at position 252; and / or (b) threonine (T) at position 254; and / or (c) at position 256. Including glutamic acid (E); where the amino acid numbering is according to the EU index described in Kabat.

  Also provided is a method of reducing the Fc-inducible effector function of a parent polypeptide comprising an Fc domain and increasing its half-life, wherein the method comprises (a) replacing the amino acid at position 234 in the Fc domain with phenylalanine (F) (B) replacing the amino acid at position 235 in the Fc domain with alanine (A), asparagine (N), phenylalanine (F), glutamine (Q), or valine (V); and (c) Replacing the amino acid at position 322 of the Fc domain with alanine (A), aspartic acid (D), glutamic acid (E), histidine (H), asparagine (N), or glutamine (Q); or position 331 of the Fc domain Substituting the amino acids of alanine (A) or glycine (G); And (d) substituting the amino acid at position 252 with tyrosine (Y) or serine (S) or tryptophan (W) or threonine (T); wherein the amino acid numbering of the Fc domain is as described in Kabat Follow the EU index. In one embodiment of the above method, the amino acid at position 252 is substituted with tyrosine (Y), where the amino acid numbering is according to the EU index set forth in Kabat. In certain embodiments of the above method, (a) the amino acid at position 254 can be replaced with threonine (T); and (b) the amino acid at position 256 is glutamic acid (E) or serine (S), or arginine (R). Or glutamine (Q), where the amino acid numbering of the Fc domain follows the EU index set forth in Kabat. In another embodiment of the above method, the amino acid at position 256 is substituted with glutamic acid (E), where the amino acid numbering is according to the EU index described in Kabat. In certain embodiments of the above methods, the amino acid at position 234 is replaced with phenylalanine (F); the amino acid at position 235 is replaced with glutamine (Q); the amino acid at position 322 is replaced with glutamine (Q), Amino acid numbering is according to the EU index described in Kabat. In some embodiments, the amino acid at position 234 is replaced with phenylalanine (F); the amino acid at position 235 is replaced with glutamine (Q); the amino acid at position 331 is replaced with glycine (G), where the amino acid numbering is Follows the EU index described in Kabat. In one embodiment of the above method, the amino acid at position 234 is replaced with phenylalanine (F); the amino acid at position 235 is replaced with alanine (A); the amino acid at position 322 is replaced with glutamine (Q), Amino acid numbering is according to the EU index described in Kabat. In another aspect of the above method, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC). In certain embodiments of the above methods, the effector function is complement dependent cytotoxicity (CDC). In another aspect of the above method, the polypeptide exhibits increased thermal stability compared to the same polypeptide comprising a FES-YTE IgG Fc domain. In certain embodiments of the above method, thermal stability is measured by differential scanning calorimetry (DSC). In some embodiments of the above method, the increase in thermal stability is at least 4 ° C. In a particular embodiment of the above method, thermal stability is measured by differential scanning fluorimetry (DSF) using a DSF fluorescent probe. In one embodiment of the above method, the DSF fluorescent probe is Sypro Orange. In certain embodiments of the above method, the increase in thermal stability is at least 5 ° C. In certain embodiments of the above methods, the polypeptide exhibits an increase in apparent solubility as measured using a polyethylene glycol (PEG) precipitation assay as compared to the same polypeptide comprising a FES-YTE IgG Fc domain. In another aspect of the above method, the polypeptide exhibits an increase in stability as measured using an accelerated stability assay compared to the same polypeptide comprising a FES-YTE IgG Fc domain. In certain embodiments of the above methods, the accelerated stability assay comprises (i) incubation of the polypeptide for an extended period of time, and (ii) incubation at an elevated temperature. In certain embodiments of the above methods, the accelerated stability assay comprises incubation at a high concentration. In some embodiments of the above method, the long period is at least one month. In certain embodiments of the above methods, the high concentration is at least 25 mg / ml. In certain aspects of the above method, the elevated temperature is at least 40 ° C.

FIG. 5 shows differential scanning calorimetry (DSC) thermograms of Ab4 antibodies containing wild type (Wt) Fc domains and Ab4 antibodies containing YTE and FES-YTE mutant Fc domains. The positions of the denaturation peaks corresponding to the Fab region of the antibody and the CH2 and CH3 regions are indicated. FIG. 5 shows a differential scanning calorimetry (DSC) thermogram of Ab4 antibody containing FQG-YTE, FQQ-YTE, and FAQ-YTE mutant Fc domains. The positions of the denaturation peaks corresponding to the Fab region of the antibody and the CH2 and CH3 regions are indicated. ADCC measured by cytotoxicity assay is shown. The antibody sample corresponds to Ab3 containing a wild type (WT) Fc domain, and Ab3 antibody containing a FQQ-YTE, FAQ-YTE, YTE, or FES-YTE variant Fc domain. A negative control was also used. CDC measured by cytotoxicity assay is shown. The antibody sample corresponds to Ab3 containing a wild type (WT) Fc domain, and Ab3 antibody containing FQQ-YTE, FQG-YTE, FAQ-YTE, YTE, or FES-YTE variant Fc domains. A negative control was also used. 2 shows an isoelectric focusing (IEF) gel. The antibody sample corresponds to Ab4 containing a wild type (WT) Fc domain and an Ab4 antibody containing a YTE, FES-YTE, FE-YTE, FAQ-YTE, FQG-YTE, or FQQ-YTE variant Fc domain. An IgG4 control is also included.

  The present disclosure provides a recombinant polypeptide comprising a variant Fc domain having an amino acid substitution that provides a desired property, such as reduced effector function, and improved plasma half-life, while maintaining stability, eg, thermal stability About. The disclosure specifically binds to an IgG Fc domain (eg, a human IgG Fc domain) or FcRn (preferably an Fc or hinge-Fc domain) comprising one or more amino acid modifications relative to wild-type IgG. With respect to polypeptides containing fragments thereof, and more specifically to immunoglobulins, where such modifications reduce or eliminate effector function.

In certain aspects, the present disclosure relates specifically to the modification of human or humanized IgG and other bioactive molecules comprising the FcRn binding portion of the human IgG Fc domain, which has particular use in therapy, prevention and diagnosis. . In certain embodiments, the polypeptide includes an IgG Fc domain, or an FcRn (preferably an Fc or hinge-Fc domain) that includes a modification that eliminates effector function, as well as a modification that increases the plasma half-life of the polypeptide. Contains fragments.
Definitions The term “a” or “an” entity refers to one or more entities; for example, “a polypeptide sequence” refers to one or more polypeptide sequences. Is understood to represent Thus, the terms “a” (or “an”), “one or more”, and “at least one” can be used interchangeably herein. Further, “and / or” as used herein should be understood as a specific disclosure of either or both of each of the two specified features or components. Thus, when used herein with the phrase “A and / or B”, the term “and / or” refers to “A and B”, “A or B”, “A” (alone), and “B "(Single). Similarly, when used in phrases such as “A, B, and / or C”, the term “and / or” is intended to encompass each of the following cases: A, B, and C A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed. , 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed. , 1999, Academic Press; and Oxford Dictionary of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provides those of ordinary skill in the art with many of the terms used in this disclosure.

  Units, prefixes, and symbols are displayed in a format that is accepted by System International de Units (SI). The numerical range includes a numerical value indicating the range. Unless otherwise noted, amino acid sequences are written left to right in amino to carboxy orientation. The headings described herein are not intended to limit the various aspects, which can be expressed by reference to the entire specification. Accordingly, the terms defined immediately below are defined in more detail by reference to the entire specification.

  Where an aspect is described herein as "comprising", other similar aspects described in the terms "consisting of" and / or "consisting essentially of" are also provided. It will be understood.

  Amino acids are described herein either by their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides are also described by the generally accepted single letter code.

  As used herein, the term “polypeptide” refers to a molecule composed of monomers (amino acids) linked linearly by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain or chains of two or more amino acids, and does not refer to a product of a particular length. As used herein, “protein” is intended to include molecules consisting of one or more polypeptides, and in certain embodiments can be linked by bonds other than amide bonds. On the other hand, the protein may be a single polypeptide chain. In the latter case, a single polypeptide chain may in some embodiments comprise two or more polypeptide subunits that are fused together to form a protein. The terms “polypeptide” and “protein” also include, but are not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or unnatural amino acids. Also refers to the product of post-expression modifications such as modification by. A polypeptide or protein can be derived from a natural biological source or can be produced by recombinant techniques, but is not necessarily translated from a designated nucleic acid sequence. This can be made by any method, including chemical synthesis.

  An “isolated” polypeptide, antibody, polynucleotide, vector, cell, or composition is a polypeptide, antibody, polynucleotide, vector, cell, or composition in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells or compositions include those purified to the extent that they are no longer found in nature. In certain embodiments, the isolated antibody, polynucleotide, vector, cell, or composition is substantially pure.

  A “recombinant” polypeptide or protein refers to a polypeptide or protein produced by recombinant DNA technology. A recombinantly produced polypeptide or protein expressed in a host cell can be isolated, fractionated, or partially or substantially purified by any suitable technique, as well as a natural or recombinant polypeptide of the present disclosure. Considered isolated for purposes.

  Polypeptide fragments, variants, or derivatives, and any combinations thereof are also included in the present disclosure. When referring to the polypeptides and proteins of the present disclosure, the term “fragment” encompasses any polypeptide or protein that retains at least some of the properties of a reference polypeptide or protein. Polypeptide fragments include proteolytic fragments and deletion fragments.

  As used herein, the term “variant” refers to a polypeptide sequence that differs from that of the parent polypeptide sequence by at least one amino acid modification. The parent polypeptide may be a natural polypeptide, ie, a “wild type” (WT) polypeptide, or a modified form of a wild type polypeptide. The term mutant polypeptide may refer to the polypeptide itself, a composition comprising the polypeptide, or the amino sequence that encodes it. Preferably, a variant polypeptide (eg, a polypeptide comprising a variant IgG Fc domain) has at least one amino acid modification compared to the parent polypeptide, eg, about 1 to about 10 compared to the parent polypeptide. Amino acid modifications, preferably from about 1 to about 6 amino acid modifications. Variant polypeptide sequences herein generally have at least 90% sequence identity, most commonly at least about 95% sequence identity with the parent polypeptide sequence.

  Variants of the polypeptides or proteins of the present disclosure include fragments as described above, as well as polypeptides or proteins comprising an amino acid sequence modified by amino acid substitution, deletion, or insertion. Variants may be natural or non-natural. Non-naturally occurring variants can be generated using mutagenesis techniques known in the art. Variant polypeptides can include conservative or non-conservative amino acid substitutions, deletions or additions.

  When used with a polypeptide or protein, the term “derivative” refers to a polypeptide or protein that has been modified to exhibit other characteristics not found in the native polypeptide or protein. An example of a “derivative” of a variant Fc domain is a fusion or conjugate with a second polypeptide or another molecule (eg, PEG, chromophore, or fluorophore) or atom (eg, a radioisotope). is there.

  As used herein, the term “polynucleotide” or “nucleotide” is intended to encompass a single nucleic acid and a plurality of nucleic acids, and is an isolated nucleic acid molecule or construct, such as a messenger RNA (mRNA) or Refers to plasmid DNA (pDNA). In certain embodiments, the polynucleotide comprises a conventional phosphodiester bond or a special bond (eg, an amide bond as present in peptide nucleic acids (PNA)).

  The term “nucleic acid” refers to any one or more nucleic acid segments present in a polynucleotide, such as DNA or RNA. The term “isolated” when used with a nucleic acid or polynucleotide refers to a nucleic acid molecule, DNA or RNA removed from its natural environment, eg, a polypeptide comprising a variant Fc domain contained in a vector Recombinant polynucleotides encoding are considered isolated for the purposes of this disclosure. Another example of an isolated polynucleotide includes a recombinant polynucleotide maintained in a heterologous host cell or purified (partially or substantially) from other polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the polynucleotides of this disclosure. Isolated polynucleotides or nucleic acids of the present disclosure further include such molecules produced synthetically. In addition, the polynucleotide or nucleic acid may contain regulatory elements such as promoters, enhancers, ribosome binding sites, or transcription termination signals.

  As used herein, the term “host cell” refers to a cell or a population of cells that carry or can carry a recombinant nucleic acid. The host cell may be a prokaryotic cell (eg, E. coli) or the host cell may be a eukaryotic cell, eg, a fungal cell (eg, Saccharomyces cerevisiae, Pichia pastoris ( Yeast cells such as Pichia pastoris, or Schizosaccharomyces pombe), and insect cells (eg Sf-9) or mammalian cells (eg HEK293F, CHO, COS-7, NIH-3T3) It may be.

  The present disclosure also encompasses polypeptides comprising variant IgG Fc domains that contain one or more conservative amino acid substitutions. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, such as basic side chains (eg lysine, arginine, histidine), acidic side chains (eg aspartic acid, Glutamic acid), uncharged polar side chains (eg glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) , Β-branched side chains (eg threonine, valine, isoleucine) and aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine). Thus, if an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, this substitution is considered conservative. In another aspect, a chain of amino acids can be conservatively substituted with a structurally similar chain that differs in the order and / or composition of side chain family members.

  The term “% sequence identity” between two polynucleotide or polypeptide sequences is compared to allow for additions or deletions (ie gaps) that must be introduced into the optimal alignment of the two sequences. Refers to the number of identical matched positions shared by the sequence across the window. A matched position is any position where the same nucleotide or amino acid is presented in both the subject and the reference sequence. Since gaps are not nucleotides or amino acids, gaps presented in the subject sequence are not counted. Similarly, the nucleotides or amino acids of the subject sequence are counted, not the nucleotides or amino acids from the reference sequence, so the gaps presented in the reference sequence are not counted.

  The percent sequence identity is calculated as follows: determine the number of positions where the same amino acid residue or nucleobase is present in the two strands to obtain the number of matching positions; Divide the number of matched positions by the total number of positions in the comparison window and multiply the quotient by 100 to obtain the percent sequence identity. Comparison of sequences between two sequences and determination of sequence identity (%) can be accomplished using software readily available both online and for download. Suitable software programs can obtain suitable software programs for alignment of both proteins and nucleotide sequences from various sources. One suitable program for determining sequence identity (%) is bl2seq, which is the US Government's National Center for Biotechnology Information BLAST website (blast.ncbi.nlm.nih.nih.com). part of the BLAST suite of programs available from gov). Bl2seq performs a comparison of two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs, European Bioinformatics Institute (EBI) (www.ebi.ac.uk/ (Tools / psa).

  Various regions within a single polynucleotide or polynucleotide subject sequence that are aligned with a polynucleotide or polypeptide reference sequence may each have a unique sequence identity (%). Note that sequence identity percentage values are rounded to the first decimal place. For example, 80.11, 80.12, 80.13, and 80.14 are rounded to 80.1, and 80.15, 80.16, 80.17, 80.18, and 80.19 are 80. Round to .2. Also note that the length value is always an integer.

  One of ordinary skill in the art is not limited to binary sequence-sequence comparisons driven solely by primary sequence data, generating sequence alignments for calculating percent sequence identity. A sequence alignment can be obtained from a multiple sequence alignment. One suitable program for generating multiple sequence alignments is ClustalW2, www. christal. org. Another suitable program is MUSCLE, www. drive5. com / muscle /. Alternatively, ClustalW2 and MUSCLE are also available from EBI, for example.

  Sequence alignment also integrates sequence data with data from heterogeneous sources such as structural data (eg, crystallographic protein structure), functional data (eg, location of mutations), or phylogenetic data. It will also be appreciated that can be generated. A suitable program for integrating heterogeneous data to generate multiple sequence alignments is T-Coffee, which is available at www. tcoffee. for example, from EBI, for example. It will also be appreciated that the final alignment used to calculate sequence identity (%) may be managed either automatically or manually.

  The term “antibody” recognizes a target such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combination thereof via at least one antigen recognition site within the variable region of an immunoglobulin molecule. And means an immunoglobulin molecule that specifically binds to a target. As used herein, the term “antibody” includes: intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (Fab, Fab ′, F (ab ′) 2, and Fv fragments). Single-chain Fv (scFv) mutants, multispecific antibodies such as bispecific antibodies made from at least two intact antibodies, chimeric antibodies, humanized antibodies, human antibodies, antigenic determinants of certain antibodies As well as any other modified immunoglobulin molecule comprising an antigen recognition site, so long as the antibody exhibits the desired biological activity. Antibodies are based on the identity of their heavy chain constant domains, called α, δ, ε, γ, and μ, respectively, and are classified into five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes). ). Different classes of immunoglobulins have different known subunit structures and three-dimensional configurations. The antibody may be naked or conjugated to other molecules such as toxins, radioisotopes. “Antibodies” or “immunoglobulins” used interchangeably herein are generally used interchangeably and include whole antibodies and any antigen-binding fragment or single chain.

  As used herein, the term “IgG” refers to a polypeptide belonging to the class of antibodies substantially encoded by a recognized immunoglobulin gamma gene. In humans, this class includes IgG1, IgG2, IgG3, and IgG4. In mice, this class includes IgG1, IgG2a, IgG2b, and IgG3.

  The term “antibody-binding fragment” refers to a portion of an intact antibody and to the antigenic determining variable region of an intact antibody. In the art, the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab ′, F (ab ′) 2 and Fv fragments, linear antibodies, single chain antibodies, and multispecific antibodies formed from antibody fragments. Can be mentioned.

  The term “monoclonal antibody” refers to a single antigenic determinant, or a homogeneous antibody population involved in highly specific recognition and binding of an epitope. This is in contrast to polyclonal antibodies that typically include different antibodies directed against different antigenic determinants. The term “monoclonal antibody” includes intact full-length monoclonal antibodies and antibody fragments (such as Fab, Fab ′, F (ab ′) 2, and Fv), single chain (scFv) mutants, antibody portions. Includes both fusion proteins and any other modified immunoglobulin molecules that contain an antigen recognition site. Furthermore, “monoclonal antibody” refers to an antibody produced by any number of methods, including but not limited to, hybridomas, phage selection, recombinant expression, and by transgenic animals.

  The term “human antibody” refers to an antibody that is produced by a human or that has an amino acid sequence that matches an antibody produced by a human made using any technique known in the art. The definition of a human antibody includes an intact antibody or a full-length antibody, a fragment thereof, and / or an antibody comprising at least one human heavy and / or light chain polypeptide, eg, a mouse light chain and a human heavy chain polypeptide. Contains antibodies. The term “humanized antibody” refers to an antibody that is derived from non-human (eg, mouse) immunoglobulin, which has been modified to include minimal non-human (eg, mouse) sequence.

  The term “chimeric antibody” refers to an antibody in which the amino acid sequence of an immunoglobulin molecule is derived from more than one species. Typically, the variable regions of both light and heavy chains are variable for antibodies from one species of mammal (eg, mouse, rat, rabbit, etc.) having the desired specificity, affinity, and ability. While corresponding to a region, the constant region is homologous to the sequence of an antibody from another species (usually human) in order to avoid eliciting an immune response in that species.

  The term “EU index as described in Kabat” refers to Kabat et al. , Sequences of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) refers to the human IgG1 EU antibody numbering system. All amino acid positions referred to in this application refer to EU index positions. For example, “L234” and “EU L234” both refer to the amino acid leucine at position 234 according to the EU index described in Kabat.

  As used herein, the terms “Fc domain” and “IgG Fc domain” refer to a portion of an immunoglobulin, eg, an IgG molecule, that correlates with a crystalline fragment obtained by papain digestion of an IgG molecule. The Fc region contains the C-terminal halves of the two heavy chains of an IgG molecule linked by disulfide bonds. It has no antigen binding activity but has a carbohydrate moiety and binding sites for Fc receptors such as complement and FcRn receptors (see below). For example, the Fc domain comprises the entire second constant domain CH2 (residues at EU positions 231 to 340 of IgG1, see, eg, Table 2) and the third constant domain CH3 (residues at EU positions 341 to 447 of human IgG1, For example, see Table 3).

  Fc refers to the isolated region, or the region associated with an antibody, antibody fragment, or Fc fusion protein. Due to the recognized polymorphism at several positions in the Fc domain, such as, but not limited to, EU positions 270, 272, 312, 315, 356, and 358, the sequences presented in this application There may be some differences between the known sequences. Thus, a “wild type IgG Fc domain” or “WT IgG Fc domain” refers to any naturally occurring IgG Fc domain (ie, any allele). Myriad Fc mutants, Fc fragments, Fc variants, and Fc derivatives are described in the following references: US Pat. No. 5,624,821; US Pat. No. 5,885,573. U.S. Pat. No. 5,677,425; U.S. Pat. No. 6,165,745; U.S. Pat. No. 6,277,375; U.S. Pat. No. 5,869,046; US Pat. No. 6,121,022; US Pat. No. 5,624,821; US Pat. No. 5,648,260; US Pat. No. 6,528,624; US Pat. US Pat. No. 6,194,551; US Pat. No. 6,737,056; US Pat. No. 7,122,637; US Pat. No. 7,183,387; US Pat. 332,581 Description: US Pat. No. 7,335,742; US Pat. No. 7,371,826; US Pat. No. 6,821,505; US Pat. No. 6,180,377 US Pat. No. 7,317,091; US Pat. No. 7,355,008; US 2004/0002587; and PCT Publication Number: WO 99/058572, International Publication 2011/0169164 pamphlet and International publication 2012/006635 pamphlet.

The sequences of the heavy chains of human IgG1, IgG2, IgG3, and IgG4 are listed in several sequence databases, eg, accession numbers P01857 (IGHG1_HUMAN), P01859 (IGHG2_HUMAN), P01860 (IGHG3_HUMAN), and P01861 (IGHG1_HUROro) (database). www.uniprot.org). Tables 1-3 show the amino acid sequences of IgG heavy chains from human IgG1 (SEQ ID NO: 1), IgG2 (SEQ ID NO: 2), IgG3 (SEQ ID NO: 3) and IgG4 (SEQ ID NO: 4) according to the EU index described in Kabat. And numbering. Residues that differ between IgG subclasses are shaded, and sites of known allelic variants are indicated with an asterisk ( ^* ).

  As used herein, “variant IgG Fc domain” and “IgG Fc variant domain” include one or more amino acid substitutions, deletions, insertions or modifications introduced at any position within the Fc domain. Refers to the IgG Fc domain. In certain embodiments, the mutated IgG Fc domain is one or more that results in reduced or eliminated binding affinity for FcγR and / or Clq compared to a wild-type Fc domain that does not include one or more amino acid substitutions. Amino acid substitutions. Fc binding interactions are essential for various effector functions and downstream signaling events such as, but not limited to, antibody dependent cell mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) . Thus, in certain embodiments, a variant IgG Fc domain (eg, antibody, fusion protein or conjugate) has the same amino acid sequence but does not contain one or more amino acid substitutions, deletions, insertions or modifications. A polypeptide, eg, exhibits altered binding affinity for at least one or more Fc ligands (eg, FcγR) as compared to an unmodified Fc region comprising a natural amino acid residue at the corresponding position of the Fc region. be able to.

  The term “YTE” or “YTE mutant” refers to a set of mutations in the IgG1 Fc domain that result in increased binding to human FcRn and improve the serum half-life of the antibody with the mutation. YTE mutants contain a combination of three “YTE mutations” introduced into the heavy chain of IgG: M252Y, S254T, and T256E (numbering is according to the EU index described in Kabat). See U.S. Patent No. 7,658,921, incorporated herein by reference. YTE mutants have been found to increase the serum half-life of antibodies compared to the wild-type form of the same antibody. See, for example, Dall'Acqua et al. , J .; Biol. Chem. 281: 23514-24 (2006) and US Pat. No. 7,083,784. These documents are incorporated herein by reference in their entirety. The “Y” mutant contains only the M256Y mutation, similarly the “YT” mutation contains only M252Y and S254T, and the “YE” mutation contains only M252Y and T256E. In particular, it is contemplated that other mutations may be present at EU positions 252 and / or 256. In certain embodiments, the mutation at EU position 252 may be M252F, M252S, M252W or M252T, and / or the mutation at EU position 256 may be T256S, T256Q, T256R or T256D.

  The term “FES” or “FES mutant” is a mutation in an IgG Fc domain that results in the elimination of effector function, ie, the ability of the Fc domain to mediate antibody-dependent and complement-mediated cytotoxicity. Points to the set of In certain embodiments, the FES mutant may comprise a combination of three “FES mutations”: L234F, L235E, and P331S (numbering is according to the EU index described in Kabat). These mutations cause a significant reduction in Fc domain binding to FcγRI (CD64), FcγRIIA (CD32A), FcγRIII (CD16) and C1q. See, for example, US 2011/0059078 and Oganesan et al. See Acta Crystallographica D 64: 700-704 (2008). These documents are incorporated herein by reference in their entirety. “FE” mutants contain only the L234F and L235E mutations.

  The term “FES-YTE IgG Fc domain” refers to a wild type IgG Fc domain comprising three “FES” mutations (L234F / L235E / P331S) and three “YTE” mutations (M252Y / S254T / T256E). Here, all numbering follows the EU index described in Kabat. As shown herein, combining FES and YTE mutations (eg, in the case of a “FES-YTE” Fc domain), as compared to a corresponding polypeptide that does not include such a combination of mutations, There is a significant decrease in protein stability (see, eg, Example 1 below).

  As used herein, the term “Fc fusion” refers to a protein in which one or more polypeptides or small molecules are operably linked to an Fc domain or a variant or derivative thereof. An Fc fusion binds an Fc region of an immunoglobulin to a fusion partner (generally any protein or small molecule). The role of the non-Fc portion of the Fc fusion, i.e. the fusion partner, may be to mediate binding to the target and thus it may be functionally similar to the variable region of the antibody. .

  As used herein, the term “parent” polypeptide is used to create a variant (eg, a variant polypeptide comprising an Fc domain, such as a variant Fc domain, or a variant antibody or variant Fc fusion). A polypeptide that is subsequently modified (eg, a polypeptide comprising an Fc domain, such as a parent Fc domain or an antibody or Fc fusion). The parent polypeptide may be a naturally occurring polypeptide (eg, a wild type Fc domain) or a variant or modified form of a naturally occurring polypeptide (eg, a YTE Fc domain and / or a FES-YTE Fc domain). It may be. The term parent polypeptide may refer to the polypeptide itself, a composition comprising the parent polypeptide, or the amino acid that encodes it. Thus, as used herein, “parent Fc domain” refers to an Fc domain that is modified to create variants, and as used herein, “parent antibody” refers to an IgG variant Fc domain. An antibody that is modified to produce a mutant antibody comprising

  As used herein, the term “IgG Fc variant domain containing polypeptide” refers to a polypeptide comprising a variant IgG Fc domain as defined above.

  An “Fc variant” comprises an Fc domain and can be present alone or in the context of an antibody, Fc fusion, isolated Fc, Fc fragment, or other polypeptide. You can also. An Fc variant may refer to the Fc polypeptide itself, a composition comprising an Fc variant polypeptide, or the amino acid sequence that encodes it. The variant IgG Fc domains described herein are defined according to the amino acid modifications that make up them. For all amino acid positions mentioned herein, the numbering always follows the EU index described in Kabat. Thus, for example, M252Y is an Fc variant in which the methionine (M) at EU position 252 is replaced with tyrosine (Y) relative to the parent Fc domain. Similarly, for example, M252Y / S254T / T256E is a variant Fc having substitutions at EU positions 252 (M to Y), 254 (S to T), and 256 (T to E), respectively, relative to the parent Fc domain. Define variants. Variants can also be referred to by names depending on their final amino acid composition at the mutated EU amino acid position. For example, the M252Y / S254T / T256E mutant can be referred to as YTE. The order in which the substitutions are described is arbitrary. That is, for example, it is noted that M252Y / S254T / T256E is the same Fc as T256E / S254T / M252Y.

  As used herein, the term “Fcγ receptor” or “FcγR” refers to any member of the family of proteins that bind to the IgG antibody Fc region and are encoded by the FcγR gene. In humans, this family includes, but is not limited to: FcγRI (CD64) such as isoforms FcγRIa, FcγRIb, and FcγRIc; isoforms FcγRIIa (such as allotypes H131 and R131), FcγRIIb (FcγRIIb) -1 and FcγRIIb-2), and FcγRII (CD32) including FcγRIIc; and FcγRIIIb (CD16) including isoforms FcγRIIIa (such as allotypes V158 and F158) and FcγRIIIb (such as allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2); Is any undiscovered human FcγR or FcγR isoform or allotype. The FcγR may be derived from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), and any undiscovered mouse FcγR or FcγR isoform or Allotypes are mentioned.

  As used herein, the terms “FcRn” or “FcRn receptor” refer to the mother or primate placenta, or the fetus via the yolk sac (rabbit), and the mother from the colostrum to the newborn via the small intestine. Fc receptors known to be involved in IgG transport (“n” represents newborn). FcRn is also known to be involved in maintaining constant serum IgG levels by binding to IgG molecules and recycling them into the serum. The binding of FcRn and IgG molecules is pH dependent, with optimal binding at pH 6.0 and weak binding at pH> 7.0. Binding of IgG to FcγR can trigger effector function (eg, ADCC), whereas binding of FcRn in a pH dependent manner can increase the serum half-life of IgG antibodies. Effector function is considered undesirable for molecules with long serum half-lives.

  As used herein, the term “effector function” refers to a biochemical event that results from the interaction of an Fc domain with an Fc receptor or ligand. Effector functions include, but are not limited to, ADCC, ADCP, and CDC. As used herein, “effector cells” refers to cells of the immune system that express one or more Fc receptors and mediate one or more effector functions. The effector cells include, but are not limited to, monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer (NK) Cells, and γδ T cells, which may be derived from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys.

  The term “antibody-dependent cell-mediated cytotoxicity” or “ADCC” bound to an Fc receptor (FcR) present on certain cytotoxic cells (eg, primarily NK cells, neutrophils, and macrophages). A polypeptide comprising an Fc domain (eg, an antibody) allows the cytotoxic effector cells to specifically bind to an antigen presenting “target cell” and then kill the target cell with a cytotoxin; Refers to the form of cytotoxicity. (Hogarth et al., Nature review Drug Discovery 2012, 11: 313). In addition to antibodies and fragments thereof, Fc domains that have the ability to specifically bind to antigen-expressing target cells, such as Fc fusions and Fc-conjugated proteins, are considered capable of performing cell-mediated cytotoxicity. .

  For simplicity, cell-mediated cytotoxicity resulting from the activity of a polypeptide comprising an Fc domain is also referred to herein as ADCC activity. Certain polypeptides of the present disclosure can be assayed for their ability to mediate target cell lysis by ADCC. To assess ADCC activity, a polypeptide of interest (eg, an antibody) is added to target cells in combination with immune effector cells to cause target cell lysis. Cell lysis is generally detected by labels from lysed cells (eg, radioactive substrates, fluorescent dyes or natural intracellular proteins). Effector cells useful for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Specific examples of in vitro ADCC assays are described in Bruggemann et al. , J .; Exp. Med. 166: 1351 (1987); Wilkinson et al. , J .; Immunol. Methods 258: 183 (2001); Patel et al. , J .; Immunol. Methods 184: 29 (1995). Alternatively or in addition, the ADCC activity of the antibody of interest can be determined in vivo, eg, Clynes et al. , Proc. Natl. Acad. Sci. USA 95: 652 (1998) can also be evaluated in an animal model.

  As used herein, the term “CDC” refers to a biochemical event of complement-dependent cytotoxicity, ie, target cell destruction mediated by the complement system.

  As used herein, the term “half-life” or “in vivo half-life” refers to the biological half-life of a particular type of polypeptide of the present disclosure in the circulation of a given animal, in the amount administered to the animal. Half is represented by the time required for circulation in the animal and / or elimination from other tissues.

  As used herein, the term “subject” refers to any animal (eg, mammal) that is the recipient of a particular treatment, including but not limited to humans, non-human primates, Examples include rodents. The terms “subject” and “patient” are used interchangeably herein when referring to a human subject.

  As used herein, the term “pharmaceutical composition” is in a form such that the biological activity of the active ingredient is effective, yet unacceptably to the subject to whom the composition is administered. A formulation that does not contain another ingredient that is toxic. Such a composition may be sterile.

  An “effective amount” of a polypeptide (eg, an antibody) disclosed herein is an amount sufficient to carry out a specifically stated purpose. A “therapeutically effective amount” can be determined based on experience and in a routine manner for a stated purpose. As used herein, “therapeutically effective amount” refers to the amount of a polypeptide (eg, antibody) or other agent effective to “treat” a disease or disorder in a subject or mammal.

  As used herein, the term “label” refers to a detectable compound or composition that is conjugated directly or indirectly to a polypeptide (eg, an antibody) to produce a “labeled” polypeptide. Point to. The label can be detected by itself (eg, a radioisotope label or fluorescent label) or, in the case of an enzyme label, can catalyze chemical alteration of a detectable foreign compound or composition. .

  The terms “treat” or “treatment” or “treat” or “relieve” or “relieve” refer to both: (1) cure, slow down symptoms of a diagnosed medical condition or disorder Therapeutic measures to reduce, reduce and / or stop the progression, and (2) preventive measures to prevent and / or delay the onset of the condition or disorder of interest. Thus, those in need of treatment include those who already have a disability; those who are susceptible to the disorder; and those who seek to prevent the disorder.

The term “vector” refers to a construct that can deliver and, in some embodiments, express one or more genes of interest to a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors coupled with cationic condensing agents, DNA encapsulated in liposomes Alternatively, RNA expression vectors and specific eukaryotic cells such as producer cells can be mentioned.
Variant IgG Fc Domain In some embodiments, a variant IgG Fc domain is provided that imparts an extended serum half-life and significantly reduces or eliminates effector function significantly. These mutated IgG Fc domains can be used, for example, to conveniently modify pharmacokinetic parameters (at the same time allowing a reduction in the number of doses) while reducing unwanted and harmful ADCC and CDC activity, for example, It can be introduced into existing therapeutic antibodies.

  YTE set of mutations (corresponding to EU M252Y, EU S254T, and EU T256E substitutions) located in the CH2 region of the Fc domain (Dall'Acqua et al., J. Immunol. 169: 5171-80 2002; Dall'Acqua et al., J. Biol. Chem. 281: 23514-24 (2006)) was found to increase the antibody serum half-life of cynomolgus monkeys by improving binding to the circulating receptor FcRn at pH 6. Yes. A FES triple mutation (corresponding to the substitution set of EU L234F, EU L235E, and EU P331S), also located in the CH2 region of the Fc domain, induces ADCC or CDC by abrogating the binding of FCγR and C1q Result in antibodies that cannot (Oganesyan et al., Acta Crystallogr. D 64: 700-704 (2008)). As revealed herein, in a variant Fc domain, eg, a variant Fc domain of an antibody. Combining these mutations results in an Fc domain with reduced thermal stability compared to a wild-type parent molecule, eg, wild-type IgG1 Fc.

  As revealed herein, certain IgG Fc domain amino acid substitutions at EU positions 234, 235, and 322 or 331 (eg, L234F / L235Q / K322Q or L234F / L235Q / P331G) may result in ADCC and CDC. Significantly reduce or eliminate. When these specific IgG Fc domain amino acid substitutions at EU positions 234, 235, and 322 or 331 are combined with a YTE mutation, the resulting mutant Ig Fc domain is equivalent to ADCC and CDC as the FES-YTE mutant. While exhibiting properties, it also exhibits greatly improved thermal stability.

Accordingly, in one aspect, a polypeptide comprising a variant IgG Fc domain (eg, a variant of a human IgG Fc domain or an FcRn binding fragment thereof) is provided, which is:
(I) a phenylalanine (F) amino acid at EU position 234;
(Ii) alanine (A), asparagine (N), phenylalanine (F), glutamine (Q) or valine (V) amino acid at EU position 235, and (iii) alanine (A), aspartic acid (D) at EU position 322 ), Glutamic acid (E), histidine (H), asparagine (N), or glutamine (Q) amino acid, or (alternatively) an alanine (A) or glycine (G) amino acid at position 331.

  In one aspect, a polypeptide comprising a variant IgG Fc domain comprising a phenylalanine (F) amino acid at EU position 234, a glutamine (Q) amino acid at EU position 235, and a glutamine (Q) amino acid at EU position 322 is provided. Hereinafter, this set of mutant IgG Fc domains and amino acid substitutions is referred to as “FQQ”.

  In another aspect, a polypeptide comprising a variant IgG Fc domain comprising a phenylalanine (F) amino acid at EU position 234, a glutamine (Q) amino acid at EU position 235, and a glycine (G) amino acid at EU position 331 is provided. The Hereinafter, this set of mutant IgG Fc domains and amino acid substitutions is referred to as “FQG”.

  In yet another aspect, a polypeptide comprising a variant IgG Fc domain comprising a phenylalanine (F) amino acid at EU position 234, an alanine (A) amino acid at EU position 235, and a glutamine (Q) amino acid at EU position 322 is provided. Is done. Hereinafter, this set of mutant IgG Fc domains and amino acid substitutions is referred to as “FAQ”.

  In certain embodiments, EU positions 234, 235, and 322 or 331 include three of the amino acid substitutions disclosed above, and one or more of any one of EU positions 252, 254, or 256 A polypeptide comprising a variant IgG Fc domain further comprising an amino acid substitution is provided. Thus, in one aspect, a tyrosine (Y) amino acid at EU position 252 or a phenylalanine (F) amino acid at EU position 252 or a serine (S) amino acid at EU position 252 or a tryptophan (W) amino acid at EU position 252; A polypeptide comprising a variant IgG Fc domain further comprising a threonine (T) amino acid at EU position 252 (eg, FQQ, FQG or FAQ variant IgG Fc domain) is provided. In a specific aspect, a polypeptide comprising a variant IgG Fc domain further comprising a tyrosine (Y) amino acid at EU position 252 (eg, FQQ, FQG or FAQ variant IgG Fc domain) is provided.

  In another embodiment, a polypeptide comprising a variant IgG Fc domain further comprising a threonine (T) amino acid at EU position 254 (eg, FQQ, FQG or FAQ variant IgG Fc domain) is provided. In another aspect, glutamic acid (E) amino acid at EU position 256, or serine (S) amino acid at EU position 256, or arginine (R) amino acid at EU position 256, or glutamine (Q) amino acid at EU position 256, or A polypeptide comprising a variant IgG Fc domain further comprising an aspartate (D) amino acid at EU position 256 (eg, FQQ, FQG or FAQ variant IgG Fc domain) is provided. In a specific embodiment, a polypeptide comprising a variant IgG Fc domain (eg, FQQ, FQG or FAQ variant IgG Fc domain) further comprising a glutamic acid (E) amino acid at EU position 256 is provided.

  In certain embodiments, EU positions 234, 235, and 322 or 311 include three of the amino acid substitutions disclosed above and are replaced with two positions selected from the group consisting of EU positions 252, 254, or 256. A polypeptide is further provided. Thus, in one aspect, a variant IgG Fc domain further comprising a tyrosine (Y) amino acid at EU position 252 and a threonine (T) amino acid at EU position 254 (eg, an FQQ, FQG or FAQ variant IgG Fc domain). A polypeptide is provided.

  In another embodiment, a polypeptide comprising an IgG Fc domain (eg, FQQ, FQG or FAQ variant IgG Fc domain) further comprising a threonine (T) amino acid at EU position 254 and a glutamic acid (E) amino acid at EU position 256 is provided. Is done. In yet another aspect, a poly comprising a variant IgG Fc domain further comprising a tyrosine (Y) amino acid at EU position 252 and a glutamic acid (E) amino acid at EU position 256 (eg, FQQ, FQG or FAQ variant IgG Fc domain). A peptide is provided.

  In certain embodiments, a polypeptide comprising a variant IgG Fc domain (eg, FQQ, FQG or FAQ variant IgG Fc domain) comprising three of the amino acids disclosed above at EU positions 234, 235, and 322 or 331 Wherein the variant IgG Fc domain further comprises a tyrosine (Y) amino acid at EU position 252, a threonine (T) amino acid at EU position 254, and a glutamic acid (E) amino acid at EU position 256.

  In certain embodiments, a phenylalanine (F) amino acid at EU position 234, a glutamine (Q) amino acid at EU position 235, a glutamine (Q) amino acid at EU position 322, a tyrosine (Y) amino acid at EU position 252 and a threonine at EU position 254 ( A polypeptide comprising a T) amino acid and a variant IgG Fc domain comprising a glutamic acid (E) amino acid at EU position 256 is provided.

  In another embodiment, phenylalanine (F) at EU position 234, glutamine (Q) amino acid at EU position 235, glycine (G) amino acid at EU position 331, tyrosine (Y) amino acid at EU position 252 and threonine (EU position 254) A polypeptide comprising a T) amino acid and a variant IgG Fc domain comprising a glutamic acid (E) amino acid at EU position 256 is provided. In yet another embodiment, phenylalanine (F) at EU position 234, alanine (A) amino acid at EU position 235, glutamine (Q) amino acid at EU position 322, tyrosine (Y) amino acid at EU position 252 and threonine at EU position 254 A polypeptide comprising a (T) amino acid and a variant IgG Fc domain comprising a glutamic acid (E) amino acid at EU position 256 is provided. Accordingly, the present disclosure includes, but is not limited to, a polypeptide comprising a variant IgG Fc domain comprising FQQ and YTE mutations, and a polypeptide comprising a variant IgG Fc domain comprising FAQ and YTE mutations. Includes peptides.

  In certain embodiments, the parent polypeptide of the variant IgG Fc domain already contains one or more of the amino acids corresponding to the aforementioned substitutions. For example, the parent Fc polypeptide may include phenylalanine (F) at EU position 234, as found in IgG4. In such embodiments, modification of one or more amino acids that already contain one or more of the disclosed substitutions is not necessary.

In certain embodiments, the variant IgG Fc domain is human. In certain other embodiments, the variant IgG Fc domain is non-human. The non-human IgG Fc domain may be derived from, for example, a rodent (eg, rat or mouse), donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken. The human variant IgG Fc domain may be, for example, a subclass IgG ₁ , IgG ₂ , IgG ₃ or IgG ₄ Fc domain. When the variant IgG Fc domain is a mouse IgG Fc domain, the domain can be, for example, a subclass IgG1, IgG2a, IgG2b, or IgG3 domain.

  In certain embodiments, a polypeptide comprising a variant IgG Fc domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 9-SEQ ID NO: 32 is provided. In another aspect, a polypeptide comprising a variant IgG Fc domain composed of an amino acid sequence selected from the group consisting of SEQ ID NO: 9 to SEQ ID NO: 32 is provided. Based on the teachings described herein, one of ordinary skill in the art will appreciate that the variant IgG Fc domains set forth in SEQ ID NO: 9-SEQ ID NO: 32 exhibit one particular allelic variation. Accordingly, in certain aspects, polypeptides are provided that include various allelic variations of the variant IgG Fc domain set forth in SEQ ID NO: 9-SEQ ID NO: 32. The sites of known allelic variation are listed in Tables 1-3.

  In certain embodiments, a polypeptide comprising a variant IgG Fc domain comprising one of the amino acid sequences disclosed in Table 4 (SEQ ID NO: 37 to SEQ ID NO: 40) is provided. In another aspect, a polypeptide comprising a variant IgG Fc domain consisting of one of the amino acid sequences disclosed in Table 4 (SEQ ID NO: 37 to SEQ ID NO: 40) is provided. Based on the teachings described herein, one of ordinary skill in the art will appreciate that the variant IgG Fc domains set forth in SEQ ID NO: 37 through SEQ ID NO: 40 exhibit one specific allelic variation. Accordingly, in certain aspects, polypeptides comprising different allelic variations of the variant IgG Fc domain set forth in SEQ ID NO: 37-SEQ ID NO: 40 are provided. The sites of known allelic variation are listed in Tables 1-3.

Increased Half Life In certain embodiments, a polypeptide comprising a variant IgG Fc domain disclosed above has improved pharmacokinetic (PK) properties compared to the same polypeptide comprising a wild type IgG Fc domain. In some embodiments, examples of improved PK properties are, for example, improved FcRn receptor binding or increased half-life. A polypeptide comprising a variant IgG Fc domain described herein is more than 5 days, more than 10 days, more than 15 days, more than 20 days, more than 25 days, more than 30 days in a mammal (eg, human), It may have a half-life (eg, serum half-life) greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months, or greater than 5 months.

  Administration of a polypeptide, as increased half-life of an IgG Fc variant domain comprising a polypeptide described herein in a mammal results in a higher serum titer of the polypeptide (eg, antibody or antibody fragment) The number of times and / or the concentration of the polypeptide to be administered can be reduced.

  In a specific embodiment, an IgG Fc containing a polypeptide comprising a substitution (eg, Y, YT, YE, YTE mutation) at one or more positions selected from the group consisting of EU positions 252, 254, and 256 The mutated domain exhibits an increased half-life compared to the parent polypeptide comprising the wild type Fc domain. In other specific embodiments, a polypeptide comprising a substitution (eg, Y, YT, YE, YTE mutation) at one or more positions selected from the group consisting of EU positions 252, 254, and 256 is included. An IgG Fc variant domain further comprises FQQ, FQG or FAQ mutations and may exhibit increased half-life as well as reduced Fc effector function compared to a parent polypeptide comprising a wild-type Fc domain.

  In a specific embodiment, an IgG Fc containing a polypeptide comprising a substitution (eg, Y, YT, YE, YTE mutation) at one or more positions selected from the group consisting of EU positions 252, 254, and 256 The mutant domain has a higher affinity for FcRn at pH 6.0 than at pH 7.4 compared to the parent polypeptide comprising the wild type Fc domain. In other specific embodiments, an IgG Fc variant domain comprising a substitution (eg, Y, YT, YE, YTE mutation) at one or more positions selected from the group consisting of EU positions 252, 254, and 256 Further comprises FQQ, FQG or FAQ and exhibits a higher affinity for FcRn at pH 6.0 than pH 7.4 and reduced Fc effector function compared to a parent polypeptide comprising a wild-type Fc domain. obtain.

Binding to an Fc Receptor An IgG Fc variant domain comprising a polypeptide described herein (eg, an antibody or fragment thereof comprising a variant IgG Fc domain) comprises at least one amino acid residue located in the Fc region. Substitutions may further be included, where such substitutions result in a reduction or elimination of affinity for at least one Fc ligand. As noted above, wild-type Fc domains interact with several ligands such as, but not limited to, FcγR receptors (eg, FcγRIIb, FcγRIIIa) and complement protein C1q, and these interactions are: It is essential for a variety of effector functions and downstream signaling events such as, but not limited to, antibody dependent cell mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). Thus, in certain embodiments, the affinity for an Fc ligand responsible for promoting effector function compared to a molecule having the same amino acid sequence as the disclosed molecule but not including substitution of at least one amino acid residue in the Fc region. IgG Fc variant domain containing polypeptides with reduced or eliminated sex are provided.

  In certain embodiments, IgG Fc variant domain-containing polypeptides are provided that include one or more of the following properties: reduction or elimination of effector function (ADCC and / or CDC), reduction of binding to Fc receptors. Or removal, or reduction or elimination of cytotoxicity. In certain aspects, IgG Fc variant domain containing polypeptides that exhibit reduced affinity for FcγR receptors (eg, FcγRIIb, FcγRIIIa) and / or complement protein C1q are provided. In some aspects, the IgG Fc variant domain-containing polypeptide has increased binding to an FcRn receptor.

One skilled in the art will appreciate that IgG Fc variant domain-containing polypeptides may have altered FcγR and / or C1q binding properties. Examples of binding properties include, but are not limited to, binding specificity, equilibrium dissociation constant (K _D ), dissociation and association constants (k _off and k _on, respectively), binding affinity and / or binding activity. In the art, the equilibrium dissociation constant (K _D ) is defined as k _off / k _on .

One skilled in the art can determine which kinetic parameters are most important for a given therapeutic or diagnostic application. For example, modifications that reduce binding to one or more positive regulators (eg, FcγRIIIA) and / or increased binding to inhibitory Fc receptors (eg, FcγRIIB) may reduce ADCC activity. Is preferred. Thus, the binding affinity of the ratio (e.g., K _D) can indicate whether ADCC activity of the IgG Fc variant domain containing polypeptide is increased or reduced or the. Furthermore, modifications that reduce binding to C1q are preferred in reducing or eliminating CDC activity.

  The affinity and binding properties of a variant IgG Fc domain-containing polypeptide for its ligand can be determined by various in vitro assay methods known in the art for determining Fc-FcγR interactions, ie Fc regions and specific binding (live Chemical or immunological assays). Such methods include equilibrium methods (eg, enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)), or kinetics (eg, surface plasma resonance such as BIACORE® analysis), as well as indirect Other methods include binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis and chromatography (eg, gel filtration).

  These and other methods may use and / or are not limited to a label on one or more components to be examined. Various detection methods such as these may be used. A detailed description of binding affinity and kinetics can be found in Paul, W. et al. E. , Ed. Fundamental Immunology, 4th Ed. Lippincott-Raven, Philadelphia (1999).

  In one aspect, compared to a parent polypeptide comprising a wild-type Fc domain, one or more Fc receptors, such as, but not limited to, FcγRI (such as FcγRIa, FcγRIb, and FcγRIc isoforms); FcγRII IgG Fc variant domain-containing polypeptides that exhibit reduced binding affinity for FcγRIIIa (such as FcγRIIIa and FcγRIIIb isoforms) are provided. In another aspect, the binding of the IgG Fc variant domain-containing polypeptide to one or more Fc receptors described above is completely eliminated.

  In one aspect, an IgG Fc variant domain-containing polypeptide is provided that exhibits reduced affinity for FcγRI compared to a parent polypeptide comprising a wild type Fc domain. In another aspect, at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 7-fold, or at least 10-fold, or at least 20-fold, or at least 30-fold than the parent polypeptide comprising a wild-type Fc domain. Or at least 40 times, or at least 50 times, or at least 60 times, or at least 70 times, or at least 80 times, or at least 90 times, or at least 100 times, or at least 200 times lower or reduced to undetectable levels Provided are IgG Fc variant domain-containing polypeptides that exhibit affinity for the FcγRI receptor.

  In another embodiment, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10 than the parent polypeptide comprising a wild type Fc domain. %, Or at least 5% lower, IgG Fc variant domain-containing polypeptides that exhibit affinity for the FcγRI receptor are provided. In some embodiments, the FcγRI is isoform FcγRIa. In another aspect, the FcγRI is isoform FcγRIb. In yet another aspect, the FcγRI is isoform FcγRIc.

  In one aspect, an IgG Fc variant domain-containing polypeptide is provided that exhibits reduced affinity for FcγRII as compared to a parent polypeptide comprising a wild-type Fc domain. In another aspect, at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 7-fold, or at least 10-fold, or at least 20-fold, or at least 30-fold than the parent polypeptide comprising a wild-type Fc domain. Or exhibit at least 40-fold, or at least 50-fold, or at least 60-fold, or at least 70-fold, or at least 80-fold, or at least 90-fold, or at least 100-fold, or at least 200-fold lower affinity for FcγRII receptor IgG Fc variant domain-containing polypeptides are provided.

  In another embodiment, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10 than the parent polypeptide comprising a wild type Fc domain. %, Or at least 5% lower, IgG Fc variant domain-containing polypeptides that exhibit affinity for the FcγRII receptor are provided. In some embodiments, the FcγRII is isoform FcγRIIa. In another aspect, the FcγRIIa isoform is allotype H131. In yet another aspect, the FcγRIIa isoform is allotype R131. In another aspect, the FcγRII is isoform FcγRIIb. In certain embodiments, the FcγRIIb isoform is FcγRIIb-1. In another aspect, the FcγRIIb isoform is FcγRIIb-2. In yet another aspect, the FcγRII is isoform FcγRIIc.

  In one aspect, an IgG Fc variant domain containing polypeptide is provided that exhibits reduced affinity for FcγRIII compared to a parent polypeptide comprising a wild type Fc domain. In another aspect, at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 7-fold, or at least 10-fold, or at least 20-fold, or at least 30-fold than the parent polypeptide comprising a wild-type Fc domain. Or exhibit at least 40-fold, or at least 50-fold, or at least 60-fold, or at least 70-fold, or at least 80-fold, or at least 90-fold, or at least 100-fold, or at least 200-fold lower affinity for FcγRIII receptor IgG Fc variant domain-containing polypeptides are provided.

  In another embodiment, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10 than the parent polypeptide comprising a wild type Fc domain. %, Or at least 5% lower, IgG Fc variant domain containing polypeptides exhibiting affinity for the FcγRIII receptor are provided. In some embodiments, the FcγRIII is isoform FcγRIIIa. In another aspect, the FcγRIIIa is allotype 158V (F158V allelic variant). In another aspect, the FcγRIIIa is allotype 158F. In another aspect, the FcγRIII is isoform FcγRIIIb. In another aspect, the FcγRIIIb is allotype NA1. In another aspect, the FcγRIIIb is allotype NA2.

  An IgG Fc variant domain-containing polypeptide described herein (eg, an antibody or fragment thereof comprising a variant IgG Fc domain) may further comprise a substitution of at least one amino acid residue located in the Fc region; Such substitution results in increased affinity for FcRn. As described above, the pH-dependent interaction between the wild-type Fc domain and FcRn extends the half-life. In one aspect, an IgG Fc variant domain containing polypeptide is provided that exhibits increased affinity for FcRn as compared to a parent polypeptide comprising a wild type Fc domain. In another aspect, at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 7-fold, or at least 10-fold, or at least 20-fold, or at least 30-fold than the parent polypeptide comprising a wild-type Fc domain. Or at least 40-fold, or at least 50-fold, or at least 60-fold, or at least 70-fold, or at least 80-fold, or at least 90-fold, or at least 100-fold, or at least 200-fold higher affinity for FcRn receptor IgG Fc variant domain-containing polypeptides are provided. In another aspect, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10 than the parent polypeptide comprising a wild type Fc domain. %, Or at least 5% higher, IgG Fc variant domain-containing polypeptides that exhibit affinity for FcRn receptors are provided. In certain aspects, IgG Fc variant domain-containing polypeptides that exhibit higher affinity for FcRn at pH 6.0 than at pH 7.4 are provided.

  In one aspect, about 100 nM to about 100 μM, or about 100 nM to about 10 μM, or about 100 nM to about 1 μM, or about 1 nM to about 100 μM, or about 10 nM to about 100 μM, or about 1 μM to about 100 μM, or about 10 μM to about IgG Fc variant domain-containing polypeptides that exhibit an affinity for the FcγR receptor that is 100 μM are provided. In certain aspects, IgG Fc variant domain-containing polypeptides that exhibit an affinity for FcγR receptors that are greater than 1 μM, greater than 5 μM, greater than 10 μM, greater than 25 μM, greater than 50 μM, greater than 100 μM are provided. In another aspect, an IgG Fc variant domain-containing poly that exhibits an affinity for an FcγR receptor that is less than 100 μM, less than 50 μM, less than 10 μM, less than 5 μM, less than 2.5 μM, less than 1 μM, or less than 100 nM, or less than 10 nM. A peptide is provided.

  In a specific aspect, there is provided a polypeptide comprising an FQQ, FQG or FAQ variant IgG Fc domain that exhibits reduced affinity for FcγR as compared to a parent polypeptide comprising a wild type IgG Fc domain. In another specific embodiment, the FQQ, FQG or further comprising a substitution (eg, Y, YT, YE, YTE mutation) at one or more positions selected from the group consisting of EU positions 252, 254, and 256 A polypeptide comprising a FAQ variant IgG Fc domain is provided, which exhibits reduced affinity for FcγR compared to a parent polypeptide comprising a wild type IgG Fc domain.

  In a specific aspect, there is provided a polypeptide comprising an FQQ, FQG or FAQ variant IgG Fc domain that is completely decoupled from FcγR compared to a parent polypeptide comprising a wild type IgG Fc domain. In another specific embodiment, the FQQ, FQG or further comprising a substitution (eg, Y, YT, YE, YTE mutation) at one or more positions selected from the group consisting of EU positions 252, 254, and 256 A polypeptide comprising a FAQ variant IgG Fc domain is provided, which is completely decoupled from FcγR compared to a parent polypeptide comprising a wild type IgG Fc domain.

  In a specific aspect, a polypeptide comprising an FQQ, FQG or FAQ variant IgG Fc domain that exhibits increased affinity for FcRn compared to a parent polypeptide comprising a wild type Fc domain is provided. In another specific embodiment, the FQQ, FQG, or A polypeptide comprising a FAQ variant IgG Fc domain is provided, which exhibits increased affinity for FcRn compared to a parent polypeptide comprising a wild type Fc domain.

Binding to C1q The complement activation pathway is initiated by binding of the first component of the complement system (C1q) to a molecule complexed with a cognate antigen, eg, an IgG Fc variant domain containing polypeptide. To assess complement activation, see, for example, Gazzano-Santoro et al. , J .; Immunol. CDC assays as described in Methods, 202: 163 (1996) can be performed.

  In one aspect, an IgG Fc variant domain-containing polypeptide is provided that exhibits reduced affinity for C1q as compared to a parent polypeptide comprising a wild type IgG Fc domain. In another aspect, at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 7-fold, or at least 10-fold, or at least 20-fold, or at least 30-fold than the parent polypeptide comprising a wild-type IgG Fc domain. , Or at least 40-fold, or at least 50-fold, or at least 60-fold, or at least 70-fold, or at least 80-fold, or at least 90-fold, or at least 100-fold, or at least 200-fold lower affinity for C1q receptor IgG Fc variant domain-containing polypeptides are provided.

  In another aspect, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least than the parent polypeptide comprising a wild type IgG Fc domain. An IgG Fc variant domain-containing polypeptide that exhibits 10%, or at least 5% lower affinity for C1q is provided.

  In another embodiment, from about 100 nM to about 100 μM, or from about 100 nM to about 10 μM, or from about 100 nM to about 1 μM, or from about 1 nM to about 100 μM, or from about 10 nM to about 100 μM, or from about 1 μM to about 100 μM, or from about 10 μM IgG Fc variant domain-containing polypeptides that exhibit an affinity for C1q that is about 100 μM are provided. In certain aspects, IgG Fc variant domain-containing polypeptides are provided that exhibit an affinity for C1q that is greater than 1 μM, greater than 5 μM, greater than 10 μM, greater than 25 μM, greater than 50 μM, greater than 100 μM.

  In a specific aspect, there is provided a polypeptide comprising an FQQ, FQG or FAQ variant IgG Fc domain that exhibits reduced affinity for C1q as compared to a parent polypeptide comprising a wild type IgG Fc domain. In another specific embodiment, the FQQ, FQG or further comprising a substitution (eg, Y, YT, YE, YTE mutation) at one or more positions selected from the group consisting of EU positions 252, 254, and 256 A polypeptide comprising a FAQ variant IgG Fc domain is provided, which exhibits reduced affinity for C1q compared to a parent polypeptide comprising a wild type IgG Fc domain.

  Certain embodiments provide a polypeptide comprising an FQQ, FQG or FAQ variant IgG Fc domain that is completely decoupled from C1q as compared to a parent polypeptide comprising a wild type IgG Fc domain. . In another specific embodiment, the FQQ, FQG or further comprising a substitution (eg, Y, YT, YE, YTE mutation) at one or more positions selected from the group consisting of EU positions 252, 254, and 256 A polypeptide comprising a FAQ variant IgG Fc domain is provided, which is completely decoupled from C1q compared to a parent polypeptide comprising a wild type IgG Fc domain.

Reduced ADCC activity In one aspect, an IgG Fc variant domain-containing polypeptide is provided that exhibits reduced ADCC activity compared to a parent polypeptide comprising a wild type IgG Fc domain. In another aspect, exhibits ADCC activity that is at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 10-fold, or at least 50-fold, or at least 100-fold less than the parent polypeptide comprising a wild-type IgG Fc domain. Or an IgG Fc variant domain-containing polypeptide having undetectable ADCC activity at a concentration of 300 μg / ml. In yet another aspect, at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60% relative to the parent polypeptide comprising a wild type IgG Fc domain, or IgG Fc variant domains that exhibit reduced ADCC activity by at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 200%, or at least 300%, or at least 400%, or at least 500% Containing polypeptides are provided. In certain embodiments, IgG Fc variant domain-containing polypeptides having undetectable ADCC activity are provided.

  In a specific aspect, the reduction or elimination of ADCC activity may be due to the reduced affinity that the IgG Fc variant domain-containing polypeptides described herein exhibit for Fc ligands and / or receptors. .

  In a specific embodiment, a polypeptide comprising an FQQ, FQG or FAQ variant IgG Fc domain that exhibits reduced or eliminated ADCC activity compared to a parent polypeptide comprising a wild type IgG Fc domain is provided. In another specific embodiment, the FQQ, FQG or further comprising a substitution (eg, Y, YT, YE, YTE mutation) at one or more positions selected from the group consisting of EU positions 252, 254, and 256 A polypeptide comprising a FAQ variant IgG Fc domain is provided, which exhibits reduced or eliminated ADCC activity compared to a parent polypeptide comprising a wild type IgG Fc domain.

  The IgG variant domain-containing polypeptides described herein can be characterized by an in vitro functional assay to determine one or more FcγR-mediated effector cell functions. In certain aspects, IgG Fc variant domain-containing polypeptides are provided that have similar binding properties and effector cell functions in an in vivo model as in in vitro assays. However, the present disclosure does not exclude IgG Fc variant domain containing polypeptides that do not exhibit the desired phenotype in in vitro assays, but do exhibit the desired phenotype in vivo.

Reduced CDC activity In one aspect, an IgG Fc variant domain-containing polypeptide is provided that exhibits reduced CDC activity compared to a parent polypeptide comprising a wild-type IgG Fc domain. In another aspect, exhibits CDC activity that is at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 10-fold, or at least 50-fold, or at least 100-fold less than the parent polypeptide comprising a wild-type IgG Fc domain. An IgG Fc variant domain-containing polypeptide is provided. In yet another aspect, at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60% relative to the parent polypeptide comprising a wild type IgG Fc domain, or IgG Fc variant domains that exhibit reduced CDC activity by at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 200%, or at least 300%, or at least 400%, or at least 500% Containing polypeptides are provided. In certain embodiments, IgG Fc variant domain-containing polypeptides that do not exhibit any detectable CDC activity are provided. In a specific embodiment, the reduction and / or elimination of CDC activity may be due to the reduced affinity that IgG Fc variant domain-containing polypeptides exhibit for Fc ligands and / or receptors.

  In specific embodiments, polypeptides comprising FQQ, FQG or FAQ variant IgG Fc domains that exhibit reduced or complete elimination of CDC activity compared to a parent polypeptide comprising a wild type IgG Fc domain are provided. In another specific embodiment, the FQQ, FQG or further comprising a substitution (eg, Y, YT, YE, YTE mutation) at one or more positions selected from the group consisting of EU positions 252, 254, and 256 A polypeptide comprising a FAQ variant IgG Fc domain is provided, which exhibits reduced or complete elimination of CDC activity compared to a parent polypeptide comprising a wild type IgG Fc domain.

Reduced toxicity Effector functions (eg, ADCC and CDC) can be an important mechanism contributing to clinical efficacy, but biological therapies are directed at the immune system to recognize and attack unwanted cells and / or targets. It is understood in the art that it can present harmful toxicity challenges related to the complex nature of commanding. Furthermore, if attack recognition and / or targeting is not performed at the site where treatment is needed, consequences such as harmful toxicity can occur. Thus, depending on the desired mechanism of action, the effector function of a given therapeutic molecule can be adjusted to reduce the associated toxicity.

  In one aspect, an IgG Fc variant domain containing polypeptide is provided that exhibits reduced toxicity as compared to a parent polypeptide comprising a wild type IgG Fc domain. In another aspect, at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 7-fold, or at least 10-fold, or at least 20-fold, or at least 30-fold than the parent polypeptide comprising a wild-type IgG Fc domain. Or an IgG Fc variant that exhibits at least 40-fold, or at least 50-fold, or at least 60-fold, or at least 70-fold, or at least 80-fold, or at least 90-fold, or at least 100-fold, or at least 200-fold lower toxicity Domain-containing polypeptides are provided. In another aspect, at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least relative to a parent polypeptide comprising a wild type IgG Fc domain IgG Fc variant domain-containing poly that exhibits reduced toxicity by 70%, or at least 80%, or at least 90%, or at least 100%, or at least 200%, or at least 300%, or at least 400%, or at least 500% A peptide is provided.

  In a specific embodiment, a polypeptide comprising an FQQ, FQG or FAQ variant IgG Fc domain that exhibits reduced toxicity as compared to a parent polypeptide comprising a wild type IgG Fc domain is provided. In another specific embodiment, the FQQ, FQG or further comprising a substitution (eg, Y, YT, YE, YTE mutation) at one or more positions selected from the group consisting of EU positions 252, 254, and 256 A polypeptide comprising a FAQ variant IgG Fc domain is provided, which exhibits reduced toxicity compared to a parent polypeptide comprising a wild type IgG Fc domain.

Increased Stability In another aspect, an IgG Fc variant domain-containing polypeptide is provided that has increased stability compared to the same polypeptide comprising a FES-YTE variant IgG Fc domain, eg, thermal stability. The In some embodiments, the FQQ, FQG, or FAQ variant further comprises a substitution (eg, Y, YT, YE, YTE mutation) at one or more positions selected from the group consisting of EU positions 252, 254, and 256. A polypeptide comprising an IgG Fc domain is provided, which has increased stability, eg, thermal stability, compared to the same polypeptide comprising a FES-YTE variant IgG Fc domain. In a specific aspect, a polypeptide comprising an FQQ, FQG or FAQ variant IgG Fc domain further comprising a YTE mutation is provided, as compared to the same polypeptide comprising a FES-YTE variant IgG Fc domain, Has increased stability, eg, thermal stability.

  As used herein, the term “stability” refers to an art-recognized measure of maintenance of one or more physical properties of a polypeptide in response to environmental conditions (eg, high or low temperature). In certain aspects, the physical property may be maintenance of the covalent structure of the polypeptide (eg, absence of proteolytic cleavage, unwanted oxidation or deamination). In another aspect, the physical property may also be the presence of the polypeptide in a properly folded state (eg, the absence of soluble or insoluble aggregates or precipitates). In one aspect, the stability of a polypeptide is determined by the biophysical properties of the polypeptide, eg, thermal instability, pH unfolding profile, stable removal of glycosylation, biochemical function (eg, binding to another protein Capacity), fragmentation rate (%), loss of purity, etc., and / or combinations thereof. In another aspect, the biochemical function is evidenced by the binding affinity of the interaction.

  In one aspect, the measure of protein stability is thermal stability, ie resistance to thermal attack. Stability can be measured using methods known in the art, such as HPLC (High Performance Liquid Chromatography), SEC (Size Exclusion Chromatography), DLS (Dynamic Light Scattering) and the like. Methods for measuring thermal stability include, but are not limited to, differential scanning calorimetry (DSC), differential scanning fluorimetry (DSF), circular dichroism (CD), and thermal attack assay. .

  In certain embodiments, an IgG Fc variant domain-containing polypeptide is provided that exhibits increased thermal stability compared to the same polypeptide comprising a FES-YTE variant IgG Fc domain. In some embodiments, the thermal stability as measured by DSC is at least 1 ° C, at least 2 ° C, at least 3 ° C, at least 4 ° C, at least 5 compared to the same polypeptide comprising a FES-YTE variant IgG Fc domain. An IgG Fc variant domain-containing polypeptide that exhibits an increase of 0 ° C., at least 6 ° C., at least 7 ° C., at least 8 ° C., at least 9 ° C. or at least 10 ° C. is provided. In another aspect, the thermal stability as measured by DSC compared to the same polypeptide comprising a FES-YTE variant IgG Fc domain is about 1 ° C., about 2 ° C., about 3 ° C., about 4 ° C., about IgG Fc variant domain-containing polypeptides that exhibit an increase of 5 ° C., about 6 ° C., about 7 ° C., about 8 ° C., about 9 ° C. or about 10 ° C. are provided.

  In some embodiments, the FQQ, FQG, or FAQ variant further comprises a substitution (eg, Y, YT, YE, YTE mutation) at one or more positions selected from the group consisting of EU positions 252, 254, and 256. A polypeptide comprising an IgG Fc domain is provided, which exhibits an increase in thermal stability as measured by DSC compared to the same polypeptide comprising a FES-YTE variant IgG Fc domain. In a specific aspect, a polypeptide comprising a FQQ, FQG or FAQ variant IgG Fc domain further comprising a YTE mutation is provided, as compared to a parent polypeptide comprising a FES-YTE variant IgG Fc domain, It exhibits an increase in thermal stability as measured by DSC.

  In certain aspects, an IgG Fc variant domain-containing polypeptide is provided that exhibits increased thermal stability compared to the same polypeptide comprising a FES-YTE variant IgG Fc domain. In some embodiments, thermal stability is measured using DSF and SYPRO® Orange DSF fluorescent probes. A person skilled in the art can measure the protein stability by DSF using a fluorescent probe other than SYPRO® Orange, such as Nile Red, SYPRO® Red, dapoxyl sulfonic acid, bis-aniline, among others. Linonaphthalenesulfonic acid (bis-ANS), 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS), or CPM (eg, Niesen et al., Nature Protocols 2: 2212-21 (2007)). It will be appreciated that (see) can be used.

  In certain embodiments, the thermal stability as measured by DSF using SYPRO® Orange is at least 1 ° C., at least 2 ° C., at least as compared to the same polypeptide comprising a FES-YTE variant IgG Fc domain. IgG Fc variant domain-containing polypeptides that exhibit an increase of 3 ° C, at least 4 ° C, at least 5 ° C, at least 6 ° C, at least 7 ° C, at least 8 ° C, at least 9 ° C, or at least 10 ° C are provided. In another aspect, compared to the same polypeptide comprising a FES-YTE variant IgG Fc domain, the thermal stability as measured by DSF using SYPRO® Orange is about 1 ° C., about 2 ° C., IgG Fc variant domain-containing polypeptides that exhibit an increase of about 3 ° C, about 4 ° C, about 5 ° C, about 6 ° C, about 7 ° C, about 8 ° C, about 9 ° C, or about 10 ° C are provided.

  In certain embodiments, an FQQ, FQG, or FAQ variant further comprising a substitution (eg, Y, YT, YE, YTE mutation) at one or more positions selected from the group consisting of EU positions 252, 254, and 256 A polypeptide comprising an IgG Fc domain is provided, which is compared to the same polypeptide comprising a FES-YTE variant IgG Fc domain, in terms of thermal stability as measured by DSF using SYPRO® Orange. Present an increase. In a specific aspect, a polypeptide comprising a FQQ, FQG or FAQ variant IgG Fc domain further comprising a YTE mutation is provided, as compared to a parent polypeptide comprising a FES-YTE variant IgG Fc domain, It presents an increase in thermal stability as measured by DSF using SYPRO® Orange.

  In certain embodiments, an IgG Fc variant domain-containing polypeptide is provided that exhibits increased apparent solubility as measured using a polyethylene glycol (PEG) precipitation assay as compared to the same polypeptide comprising a FES-YTE IgG Fc domain. Is done. See, eg, Middaugh et al. , J .; Biol. Chem. 254: 367-370 (1979); Shire et al. , Eds. , 2010, Current Trends in Monoclonal Antibody Development and Manufacturing, Springer; Gibson et al. , J .; Pharm. Sci. 100: 1009-21 (2011). In some embodiments, the FQQ, FQG, or FAQ variant further comprises a substitution (eg, Y, YT, YE, YTE mutation) at one or more positions selected from the group consisting of EU positions 252, 254, and 256. A polypeptide comprising an IgG Fc domain is provided, which has an increased apparent solubility measured using a polyethylene glycol (PEG) precipitation assay compared to the same polypeptide comprising a FES-YTE variant IgG Fc domain. Present. In a specific aspect, a polypeptide comprising a FQQ, FQG or FAQ variant IgG Fc domain further comprising a YTE mutation is provided, as compared to a parent polypeptide comprising a FES-YTE variant IgG Fc domain, It exhibits increased apparent solubility as measured using a polyethylene glycol (PEG) precipitation assay.

  Biopharmaceuticals in storage change over time, but are considered stable as long as their properties are maintained within the manufacturer's specifications. The number of days that the formulation remains stable at the recommended storage conditions is called shelf life. An experimental protocol commonly used for data collection that serves as a basis for estimating the shelf life of a formulation is called a stability assay. Shelf life is generally estimated according to the type of stability test: real-time stability assay and accelerated stability assay. In an accelerated stability assay, the formulation is stored under increased stress conditions: eg temperature, humidity and pH. For example, Tydeman and Kirkwood, J. et al. Biol. Stand. 12: 195-206 (1984); Some et al. , J .; Pharm. Sci. 90: 1759-66 (2001); FDA. Guidelines for submitting documents for the stability of human drugs and biologics. See Rockville (MD), 1987.

  In certain embodiments, an IgG Fc variant domain-containing polypeptide is provided that exhibits increased stability as measured using an accelerated stability assay compared to the same polypeptide comprising a FES-YTE variant IgG Fc domain. The In some embodiments, the FQQ, FQG, or FAQ variant further comprises a substitution (eg, Y, YT, YE, YTE mutation) at one or more positions selected from the group consisting of EU positions 252, 254, and 256. A polypeptide comprising an IgG Fc domain is provided, which exhibits an increased stability as measured using an accelerated stability assay compared to the same polypeptide comprising a FES-YTE variant IgG Fc domain. In a specific aspect, a polypeptide comprising an FQQ, FQG or FAQ variant IgG Fc domain further comprising a YTE mutation is provided, as compared to the same polypeptide comprising a FES-YTE variant IgG Fc domain, It presents an increase in stability as measured using an accelerated stability assay.

  In certain embodiments, the accelerated stability assay comprises incubation of IgG Fc variant domain-containing polypeptides over a prolonged period and / or incubation at elevated temperatures. In another embodiment, the accelerated stability assay is performed by incubation of IgG Fc variant domain containing polypeptides at high concentrations. In certain embodiments, the measurement by accelerated stability assay is performed using high performance size exclusion chromatography (HPSEC). In other embodiments, the measurement by the accelerated stability assay is performed using dynamic light scattering (DSL). One skilled in the art will appreciate that polypeptide aggregation can be measured by various methods known in the art.

  In certain embodiments, the extended duration of the accelerated stability assay is at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, or at least 4 months. In another aspect, the extended duration of the accelerated stability assay is about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, or about 4 months.

  In certain embodiments, the concentration of the IgG Fc variant domain-containing polypeptide used in the accelerated stability assay is at least 10 mg / ml, at least 15 mg / ml, at least 20 mg / ml, at least 25 mg / ml, at least 30 mg / ml, at least 35 mg / ml. ml, at least 40 mg / ml, at least 45 mg / ml, or at least 50 mg / ml. In certain embodiments, the concentration of the IgG Fc variant domain-containing polypeptide used in the accelerated stability assay is about 10 mg / ml, about 15 mg / ml, about 20 mg / ml, about 25 mg / ml, about 30 mg / ml, about 35 mg / ml. ml, about 40 mg / ml, about 45 mg / ml, or about 50 mg / ml.

  In certain embodiments, the temperature used in the accelerated stability assay is at least 30 ° C, at least 35 ° C, at least 40 ° C, at least 45 ° C, at least 50 ° C, at least 55 ° C, or at least 60 ° C. In some embodiments, the elevated temperature used in the accelerated stability assay is about 30 ° C, about 35 ° C, about 40 ° C, about 45 ° C, about 50 ° C, about 55 ° C, or about 60 ° C.

Methods In certain embodiments, methods are provided for reducing Fc-induced effector function (eg, ADCC and / or CDC) of an IgG Fc variant domain-containing polypeptide, the method comprising: (a) an EU position in the Fc domain Replacing 234 amino acids with phenylalanine (F); (b) replacing the amino acid at EU position 235 in the Fc domain with alanine (A), asparagine (N), phenylalanine (F), glutamine (Q), or valine (V). (C) replacing the amino acid at EU position 322 of the Fc domain with alanine (A), aspartic acid (D), glutamic acid (E), histidine (H), asparagine (N), or glutamine (Q) Or an amino acid at EU position 331 of the Fc domain Comprising the step of replacing with alanine (A) or glycine (G). In certain embodiments, the Fc domain of the parent polypeptide comprises tyrosine (Y) at EU position 252; and / or threonine (T) at EU position 254; and / or glutamic acid (E) at EU position 256. In certain specific embodiments, the Fc domain of the parent polypeptide comprises tyrosine (Y) at EU position 252, threonine (T) at EU position 254, and glutamate (E) at EU position 256. That is, the Fc domain of the parent polypeptide is a YTE mutant IgG Fc domain.

  In certain embodiments, methods of reducing Fc-induced effector function (eg, ADCC and / or CDC) of a parent polypeptide comprising an IgG Fc domain and increasing its half-life are also provided: a) replacing the amino acid at EU position 234 in the Fc domain with phenylalanine (F); (b) replacing the amino acid at EU position 235 in the Fc domain with alanine (A), asparagine (N), phenylalanine (F), glutamine (Q ) Or valine (V); and (c) the amino acid at EU position 322 of the Fc domain is alanine (A), aspartic acid (D), glutamic acid (E), histidine (H), asparagine (N) Or substituting with glutamine (Q); or Fc domain Step substituting amino acids in the EU position 331 with alanine (A) or glycine (G); amino acids and (d) EU position 252 including a step of replacing tyrosine (Y). In certain aspects, the method further comprises substituting the amino acid at EU position 254 with threonine (T); and substituting the amino acid at EU position 256 with glutamic acid (E).

  In certain embodiments, methods of reducing Fc-induced effector function (eg, ADCC and / or CDC) and reducing Fc-induced effector function (eg, ADCC and / or CDC) and increasing the aforementioned half-life The method replaces the amino acid at position EU 234 in the Fc domain with phenylalanine (F); the amino acid at position 235 in the Fc domain with glutamine (Q); the amino acid at position 322 in the Fc domain with glutamine (Q) including.

  In certain embodiments, methods of reducing Fc-induced effector function (eg, ADCC and / or CDC) and reducing Fc-induced effector function (eg, ADCC and / or CDC) and increasing the aforementioned half-life The method replaces the amino acid at EU position 234 of the Fc domain with phenylalanine (F); the replacement of the amino acid at EU position 235 of the Fc domain with glutamine (Q); the replacement of the amino acid at EU position 331 of the Fc domain with glycine (G) including.

  In certain embodiments, methods of reducing Fc-induced effector function (eg, ADCC and / or CDC) and reducing Fc-induced effector function (eg, ADCC and / or CDC) and increasing the aforementioned half-life The method replaces the amino acid at position EU 234 in the Fc domain with phenylalanine (F); the amino acid at position 235 in the Fc domain with alanine (A); the amino acid at position 322 in the Fc domain with glutamine (Q) including.

Antibodies and fragments thereof In certain embodiments, an IgG Fc variant domain-containing polypeptide comprises an antigen binding domain. In certain embodiments, the antigen binding domain may be an antibody, eg, a monoclonal antibody, or an antigen binding domain thereof. The antigen binding domain may be a full length antibody, eg, a human antibody, a humanized antibody, or a chimeric antibody, or a fragment thereof. In certain embodiments, the antigen binding domain comprises, for example, a single chain antibody; a diabody; a polypeptide chain of an antibody; an F (ab ′) 2 fragment; or an F (ab) fragment.

  The term “antibody variant” refers to a variant IgG Fc domain-containing polypeptide described herein, wherein the polypeptide is an antibody. Antibody variants include monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, anti-idiotype (anti-Id) antibodies, and any of these Fc domains Fragment. In certain embodiments, antibody variants include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding domain, wherein these fragments are herein defined as It can be fused or conjugated to another immunoglobulin domain, including the variant IgG Fc domain described. In one aspect, the antibody variant is of human IgG1, IgG2, IgG3 or IgG4 isotype.

  Antibody variants and fragments thereof comprising the variant IgG Fc domains described herein are found in birds and mammals (eg, rodents such as humans, mice or rats, donkeys, sheep, rabbits, goats, guinea pigs, camels). , Horses, or chickens). In a specific aspect, antibody variants are provided that are human or humanized monoclonal antibodies. As used herein, a “human” antibody includes an antibody having the amino acid sequence of a human immunoglobulin, and also includes an antibody isolated from a mouse that expresses a human immunoglobulin or an antibody derived from a human gene.

  Antibody variants can be monospecific, bispecific, trispecific, or can have greater specificity (multispecific antibodies). Multispecific antibody variants can specifically bind to various epitopes of the desired target molecule or are specific for both the target molecule and a heterologous epitope such as a heterologous polypeptide or solid support material. Can be combined. For example, see the following references: International Publication Number: WO 94/04690 pamphlet; WO 93/17715 pamphlet; WO 92/08802 pamphlet; WO 91/00360 pamphlet; And WO 92/05793; Tutt et al. , J .; Immunol. 147: 60-69 (1991); U.S. Pat. No. 4,474,893, U.S. Pat. No. 4,714,681, U.S. Pat. No. 4,925,648, U.S. Pat. 573,920, and US Pat. No. 5,601,819; and Kostelny et al. , J .; Immunol. 148: 1547 (1992). Methods for making bispecific or multispecific antibodies are known in the art.

Methods for Producing Antibodies Containing Variant IgG Fc Domains Antibody variants or fragments thereof are produced by any method known in the art for antibody synthesis, particularly by chemical synthesis or recombinant expression techniques. be able to.

  Monoclonal antibody variants can be generated using a variety of techniques known in the art, such as the use of hybridoma, recombination, and phage display techniques, or combinations thereof. For example, monoclonal antibody variants can be generated using hybridoma technology, such as is known in the art and is taught, for example, in the following literature: Harlow et al. , Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al. , In: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981). Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art.

  Antibody variants can be generated by various methods known in the art. Non-limiting examples include isolating an antibody coding region (eg, from a hybridoma) and introducing one or more Fc domain amino acid substitutions into the isolated antibody coding region. Alternatively, the variable region can be subcloned into a vector encoding the mutant IgG Fc domain described herein.

  Antibody variant fragments that recognize specific epitopes can be generated by any technique known in the art. For some applications, such as in vivo use of antibody variants in humans and in vitro detection assays, it may be advantageous to use human or chimeric antibody variants. Completely human antibodies are particularly desirable for therapeutic treatment of human subjects. Human antibodies or fragments thereof comprising a variant IgG Fc domain described herein can be generated by various methods known in the art. For example, see the following documents: US Pat. No. 4,444,887, US Pat. No. 4,716,111; and International Publication Number: WO 98/46645, International Publication. No. 98/50433 pamphlet, WO 98/24893 pamphlet, WO 98/16654 pamphlet, WO 96/34096 pamphlet, WO 96/33735 pamphlet, and WO 91/10741. Issue brochure.

  Chimeric antibody variants or fragments thereof comprising a variant IgG Fc domain described herein can be made by a variety of methods known in the art. See, for example, the following documents: Morrison, Science 229: 1202 (1985); Oi et al. BioTechniques 4: 214 (1986); Gillies et al. , J .; Immunol. Methods 125: 191-202 (1989); US Pat. No. 5,807,715, US Pat. No. 4,816,567, US Pat. No. 4,816,397, and US Pat. No. 6,311,415. In certain aspects, the humanized antibody variant or fragment thereof comprises a variant IgG Fc domain as described herein. Humanized antibody variants can be generated using various techniques known in the art, including, but not limited to, the following: CDR grafting (European patents) No. 239,400; WO 91/09967; and US Pat. No. 5,225,539, US Pat. No. 5,530,101, and US Pat. No. 5,585, 089), veneering or resurfacing (EP 592,106 and EP 519,596; Padlan, Molecular Immunology 28: 489-498 (1991); Studnikka et al., Protein Engineering 7: 805-814 (1994); and Rogu ka et al., Proc. Natl. Acad. Sci. USA 91: 969-973 (1994)), chain shuffling (US Pat. No. 5,565,332), as well as, for example, the following references Technology: US Pat. No. 6,407,213 and US Pat. No. 5,766,886, WO 9317105, Tan et al. , J .; Immunol. 169: 1119-25 (2002), Caldas et al. , Protein Eng. 13: 353-60 (2000), Morea et al. , Methods 20: 267-79 (2000), Baca et al. , J .; Biol. Chem. 272: 10678-84 (1997), Roguska et al. , Protein Eng. 9: 895-904 (1996), Couto et al. , Cancer Res. 55 (23 Supp): 5973s-5777s (1995), Couto et al. , Cancer Res. 55: 1717-22 (1995), Sandhu, Gene 150: 409-10 (1994), and Pedersen et al. , J .; Mol. Biol. 235: 959-73 (1994).

  Human antibody variants can also be generated using transgenic mice that are unable to express functional endogenous immunoglobulin genes but are capable of expressing human immunoglobulins. For example, human heavy and light chain immunoglobulin gene complexes can be introduced into mouse embryonic stem cells randomly or by homologous recombination. Alternatively, in addition to human heavy and light chain genes, human variable regions, constant regions, and diversity regions can also be introduced into mouse embryonic stem cells. Mouse heavy and light chain immunoglobulin genes can be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and introduced into blast cells by microinjection to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring that express human antibodies. Transgenic mice are immunized with the selected antigen or immunogenic fragments thereof in the usual manner.

  Monoclonal antibodies against the antigen can be obtained from transgenic mice immunized using conventional hybridoma technology. Human immunoglobulin transgenes harbored by transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, it is possible to generate antibodies useful for therapy using such techniques. For an overview of the above techniques for generating human antibodies, see Lonberg and Huszar, Int. Res. Immunol. 13: 65-93 (1995). For details of the above-described techniques for generating human antibodies and human monoclonal antibodies and protocols for generating such antibodies, see, for example, the following documents: WO 98/24893, WO 96/34096, and WO 96/33735; and US Pat. No. 5,413,923, US Pat. No. 5,625,126, US Pat. No. 5,633,425. Specification, US Pat. No. 5,569,825, US Pat. No. 5,661,016, US Pat. No. 5,545,806, US Pat. No. 5,814,318 And US Pat. No. 5,939,598.

A polynucleotide encoding a polynucleotide IgG Fc variant domain containing polypeptide is provided. Also provided are polynucleotides that hybridize under high stringency, moderate or low stringency hybridization conditions with a polynucleotide encoding an IgG Fc variant domain-containing polypeptide.

  In certain aspects, a polynucleotide sequence encoding an IgG Fc variant domain-containing polypeptide can be generated from a parent polynucleotide sequence obtained from a suitable source. Once the polynucleotide sequence is obtained, methods known in the art for manipulation of nucleotide sequences, such as recombinant DNA techniques, site-directed mutagenesis, PCR, etc. (see, eg, techniques described in the following references: Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y ol, Proc. Et al. Sons NY; which is incorporated herein by reference in its entirety. By operating the de sequences, producing a IgG Fc variant domain containing polypeptides having different amino acid sequences, e.g., amino acid substitutions can be formed deletions, and / or insertions.

  In another aspect, a polynucleotide sequence encoding an IgG Fc variant domain-containing polypeptide can be assembled from chemically synthesized oligonucleotides (eg, Kutmejer et al. BioTechniques 17: 242 (1994)). This, in brief, involves the synthesis of overlapping oligonucleotides containing portions of the coding sequence, the annealing and ligation of these oligonucleotides, followed by amplification of the ligated oligonucleotides by PCR.

Binding molecules comprising specific antigens and fusion partner variant IgG Fc domains, such as antibodies, fusion proteins, or conjugates, can be used to target almost any molecule. In addition, almost any molecule can be incorporated into a fusion protein or conjugate comprising a variant IgG Fc domain as described herein.

  These specific target molecules and / or fusion partners include, but are not limited to, proteins belonging to the following list of proteins, as well as subunits, domains, portions and epitopes: renin; human growth hormone and bovine growth Growth hormone such as hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoprotein; α-1-antitrypsin; insulin A chain; insulin B chain; proinsulin; follicle stimulating hormone; calcitonin; Glucagon; coagulation factors such as factor VII, factor VIIIC, factor IX, tissue factor (TF), and von Willebrand factor; anticoagulation factors such as protein C; atrial natriuretic factor; lung surfactant; urokinase or human Urine or tissue plastic Plasminogen activator (t-PA) such as minogen activator; Bombesin; Thrombin; Hematopoietic growth factor; Tumor necrosis factor-α and -β; Enkephalinase; RANTES (regulated on activation normally T-cell expressed and secreted); human macrophage inflammatory protein (MIP-1-α); serum albumin such as human serum albumin; Muller tube inhibitor; relaxin A chain; relaxin B chain; prorelaxin; mouse gonadotropin related peptide; β-lactamase and other microorganisms Protein; DNase; IgE; Cytotoxic T lymphocyte associated antigen (CTLA) such as CTLA-4; Inhibin; Activin; Vascular endothelial growth factor (VEGF); Receptors for hormones or growth factors such as EGFR and VEGFR; interferons such as α interferon (α-IFN), β interferon (β-IFN) and γ interferon (γ-IFN); protein A or D; rheumatoid factor; Neurotrophic factor (BDNF), neurotrophin-3, -4, -5 or -6 (NT-3, NT-4, NT-5 or NT-6) or neurotrophic factor such as nerve growth factor; derived from platelets Growth factor (PDGF); fibroblast growth factors such as AFGF and PFGF; epidermal growth factor (EGF); TGF-α and TGF-β (TGF-1, TGF-2, TGF-3, TGF-4, or TGF- Transforming growth factors such as 5; insulin-like growth factors I and II (IGF-I and IGF) II); des (1-3) -IGF-I (brain IGF-I), insulin-like growth factor binding protein; CD2, CD3, CD4, CD8, CD11a, CD14, CD18, CD19, CD20, CD22, CD23, CD25 , CD33, CD34, CD40, CD40L, CD52, CD63, CD64, CD80 and CD147 and other CD proteins; erythropoietin; osteoinductor; immunotoxin; bone morphogenetic protein (BMP); Interferon; colony stimulating factor (CSF) such as M-CSF, GM-CSF, and G-CSF; interleukin, eg, IL-1 to IL-13; TNFα, superoxide dismutase; T cell receptor; surface membrane protein Decay accelerating factor; for example, AID Viral antigens such as parts of the envelope (eg gp120); transport proteins; homing receptors; addressins; regulatory proteins; cell adhesion molecules such as LFA-1, Mac 1, p150.95, VLA-4, ICAM-1 , ICAM-3 and VCAM, a4 / p7 integrin, and (Xv / p3 integrin (including one or more of its subunits), CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, α7, α8, Integrin α subunits such as α9, αD, CD11a, CD11b, CD51, CD11c, CD41, αIIb, αIELb; integrin β subunits such as CD29, CD18, CD61, CD104, β5, β6, β7 and β8; Not αVβ , ΑVβ5 and α4β7 combinations of integrin subunits; apoptotic pathway members; IgE; blood group antigens; flk2 / flt3 receptor; obesity (OB) receptor; mp1 receptor; CTLA-4; protein C; EphA2, EphA4 Eph receptors such as EphB2; human leukocyte antigens (HLA) such as HLA-DR; complement proteins such as complement receptors CR1, C1Rq and other complement factors such as C3 and C5; GpIbα, GpIIb / IIIa and Glycoprotein receptors such as CD200; as well as fragments of any of the polypeptides listed above.

  In certain aspects, IgG Fc variant domain-containing polypeptides (eg, antibody variants, fusion proteins, or conjugates) can specifically bind to, but are not limited to, the cancer antigens listed below: ALK receptor (playtrophin receptor), pretrophin, KS1 / 4 systemic cancer antigen; ovarian cancer antigen (CA125); prostate acid phosphate; prostate specific antigen (PSA); melanoma-associated antigen p97; melanoma antigen gp75 High molecular weight melanoma antigen (HMW-MAA); prostate specific membrane antigen; carcinoembryonic antigen (CEA); polymorphic epithelial mucin antigen; human milk fat globule antigen; CEA, TAG-72, CO17-1A, GICA19- 9, colorectal tumor-associated antigens such as CTA-1 and LEA; Burkitt lymphoma antigen-38.13 CD19; human B-cell lymphoma antigen-CD20; CD33; melanoma-specific antigens such as ganglioside GD2, ganglioside GD3, ganglioside GM2 and ganglioside GM3; tumor-specific transplanted cell surface antigen (TSTA); T antigen, DNA tumor virus and RNA Virus-induced tumor antigens such as tumor virus envelope antigens; Colon CEA, 5T4 carcinoembryonic trophoblast glycoprotein and bladder cancer tumor carcinoembryonic antigens such as tumor fetal antigen-alpha-fetoprotein; differentiation such as human lung cancer antigens L6 and L20 Antigen; fibrosarcoma antigen; human leukemia T cell antigen-Gp37; neoglycoprotein; sphingolipid; breast cancer antigen such as EGFR (epidermal growth factor receptor); NY-BR-16; NY-BR-16 and HER2 antigen ( p185HER2); polymorphism Mucin (PEM); malignant human lymphocyte antigen-APO-1; differentiation antigen such as I antigen found in fetal erythrocytes; early endoderm I antigen found in adult erythrocytes; preimplantation embryo; I (Ma) found in gastric adenocarcinoma M18, M39 found in breast epithelium; SSEA-1 found in bone marrow cells; VEP8; VEP9; My1; Va4-D5; D156-22 found in colorectal cancer; TRA-1-85 (blood group H) SCP-1 found in testis and ovarian cancer; C14 found in colon adenocarcinoma; F3 found in lung adenocarcinoma; AH6 found in gastric cancer; Y hapten; Ley found in embryonic cancer cells; TL5 (blood group) A); EGF receptor found in A431 cells; E1 series found in pancreatic cancer (blood group B); Fc10.2 found in embryonic cancer cells; gastric adenocarcinoma antigen; gland CO-514 (blood type Lea) found in adenocarcinoma; NS-10 found in adenocarcinoma; CO-43 (blood group Leb); G49 found in the EGF receptor of A431 cells; MH2 found in colon adenocarcinoma (blood Type ALeb / Ley); 19.9 found in colon cancer; gastric cancer mucin; T5A7 found in bone marrow cells; R24 found in melanoma; 4.2, GD3, D1.1, OFA-1 found in embryonic cancer , GM2, OFA-2, GD2, and M1: 22: 25: 8 and SSEA-3 and SSEA-4 found in 4-8 cell stage embryos; cutaneous T cell lymphoma antigen; MART-1 antigen; Sialy Tn (STn) antigen; colon cancer NY-CO-45; lung cancer antigen NY-LU-12 variant A; adenocarcinoma antigen ART1; Trans-tissue common antigen MA2; tumor-associated neural antigen); neuro-oncological ventral antigen 2 (NOVA2); hepatocellular carcinoma antigen gene 520; tumor-associated antigen CO- 029; tumor-associated antigen MAGE-C1 (cancer / testis antigen CT7), MAGE-B1 (MAGE-XP antigen), MAGE-B2 (DAM6), MAGE-2, MAGE-4-a, MAGE-4-b and MAGE -X2; cancer / testis antigen (NY-EOS-1) as well as a fragment of any of the polypeptides listed above.

  In yet another aspect, an IgG Fc variant domain-containing polypeptide (eg, antibody variant, fusion protein, or conjugate) can specifically bind to an infectious agent (eg, bacterium, virus). Infectious bacteria include, but are not limited to, gram negative bacteria and gram positive bacteria. Gram-positive bacteria include, but are not limited to, Pasteurella species, Staphylococci species, and Streptococcus species. Gram negative bacteria include, but are not limited to, Escherichia coli, Pseudomonas species, and Salmonella species. Specific examples of infectious bacteria include but are not limited to: Helicobacter pylori, Borrelia burgdorferi, Legionella pneumophila, Mycobacterium (Mycobacterium) species (eg, M. tuberculosis, M. avium, M. intracellulare, M. kansasiii, mycobacteria)・ Goldonae, Staphylococcus aureus, Pseudo onas aeruginosa, Neisseria gonorrhoeae, Neisseria meningacidis, Listeria monocytogenes, Streptococcus pyogenes, Streptococcus pyogenes Group Streptococcus), Streptococcus genus (Vilidans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus genus (Aerobic species), Streptococcus pneumoniae (Streptococcus) e), pathogenic Campylobacter species, Enterococcus species, Haemophilus influenzae, Bacillus anthracis species, Corynebacterium species, Corynebacterium species Erysipeperothrix rhusiopathae, Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes pneumoniae pneumoniae pneumoniae , Pasteurella multocida (Pasteurella multocida), Bacteroides (Bacteroides) species, Fusobacterium Nukureatsumu (Fusobacterium nucleatum), Streptomyces Bacillus Moniriforumisu (Streptobacillus moniliformis), Treponema pallidum (Treponema pallidum), Treponema Perutenue (Treponema pertenue), Leptospira spp. (Leptospira), Rickettsia, and Actinomyces israelii. Viruses include, but are not limited to, enteroviruses, rotaviruses, adenoviruses, and hepatitis viruses. Specific examples of viruses recognized in humans include, but are not limited to: Retroviridae (eg, Human Immunodeficiency Virus (HIV); Picoraviridae) (E.g., poliovirus, hepatitis A virus; enterovirus, human coxsackie virus, rhinovirus, echovirus); calciviridae (e.g., strain causing gastroenteritis); Togaviridae (Eg equine encephalitis virus, rubella virus); Flaviviridae (eg dengue virus, encephalitis virus, yellow fever virus); Coronaviridae (Coron) viridae) (eg, coronavirus); Rhabdoviridae (eg, vesicular stomatitis virus, rabies virus); Filoviridae (eg, Ebola virus); Paramyxoviridae (eg, Parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (eg, influenza virus), Bungaviridae (eg, Hantaan virus, Bunga virus, Flebo) Viruses (phlebovirus and Nairo virus); Arenaviridae (Arena) viridae) (haemorrhagic fever virus); reoviridae (eg, reovirus, orbivirus, and rotavirus); birnaviridae; hepadnaviridae (hepatitis B virus); Parvoviridae (Parvovirus); Papovaviridae (Papillomavirus, Polyomavirus); Adenoviridae (Most Adenovirus); Herpesviridae (eg Herpesviridae) (eg Herpesviridae) (HSV) I and II, herpes zoster virus, cytomegalovirus (CMV)); Poxviridae (decubitus virus, vaccinia Near viruses, poxviruses); Iridoviridae (eg, African swine fever virus); Pathogens; Norwalk and related viruses and astroviruses).

Conjugates and derivatives In some embodiments, variant IgG Fc domains described herein include, but are not limited to, peptides, polypeptides, proteins, fusion proteins, nucleic acid molecules, small molecules, mimetics, synthetic drugs, It can be combined or fused with one or more moieties such as inorganic molecules and organic molecules.

  In certain embodiments, an IgG Fc variant domain-containing polypeptide comprises a derivative modified, for example, by covalent attachment of any type of molecule to the polypeptide, or by chemical or enzymatic modification. For example, derivatives may be modified by, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, coupling with cellular ligands or other proteins. Contains peptides. Any of a variety of chemical modifications can be performed by known techniques, such as but not limited to specific chemical degradation, acetylation, formylation, and the like. In addition, the derivatives can also contain one or more non-classical amino acids.

  An IgG Fc variant domain-containing polypeptide can be conjugated to a polymer molecule such as a high molecular weight polyethylene glycol (PEG). PEG can be synthesized with or without a multifunctional linker, either by site-specific conjugation of PEG to the N- or C-terminus of the polypeptide, or by an epsilon-amino group present on a lysine residue. Can be conjugated to a peptide. Linear or branched polymer derivatization that minimizes loss of biological activity can be used.

  Chemically conjugated to a heterologous protein or polypeptide (or fragment thereof, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids) ( Conjugates comprising IgG Fc variant domain containing polypeptides (including both covalent and non-covalent bonds) are provided. The conjugation need not necessarily be direct, but can also be performed via a linker. Such linker molecules are known in the art and are described in the following literature: Denardo et al. Clin Cancer Res 4: 2483 (1998); Peterson et al. Bioconjug. Chem. 10: 553 (1999); Zimmerman et al. Nucl. Med. Biol. 26: 943 (1999); Garnett, Adv. Drug Deliv. Rev. 53: 171 (2002).

  Further provided are compositions comprising a heterologous protein, peptide or polypeptide conjugated to an IgG Fc variant domain containing polypeptide. Methods for fusing or linking polypeptides to antibody moieties are known in the art. For example, see the following documents: US Pat. No. 5,336,603, US Pat. Nos. 5,622,929, 5,359,046, 5,349,053. No. 5,447,851 and 5,112,946; European Patent No. 307,434, European Patent No. 367,166; 04388 and WO 91/06570; Ashkenazi et al. Proc. Natl. Acad. Sci. USA 88: 10535-39 (1991,); Zheng et al. J. et al. Immunol. 154: 5590-5600 (1995); Vil et al. Proc. Natl. Acad. Sci. USA 89: 11337-41 (1992).

  In certain embodiments, an IgG Fc variant domain containing polypeptide is conjugated to a diagnostic or detectable agent. Such conjugates can be useful in monitoring or predicting the development or progression of inflammatory diseases as part of a clinical trial procedure, such as determining the effectiveness of a particular treatment. Such diagnosis and detection can be accomplished by coupling an IgG Fc variant domain containing polypeptide with a detectable substance.

  In certain embodiments, an IgG Fc variant domain containing polypeptide is conjugated to a therapeutic agent. An IgG Fc variant domain-containing polypeptide can be conjugated to a therapeutic moiety such as a cytotoxin, eg, a static cell or cell lethal agent, a therapeutic agent or a radioactive metal ion, eg, an alpha emitter. A cytotoxin or a cell killing material includes any material that is detrimental to cells.

  In certain embodiments, an IgG Fc variant domain-containing polypeptide can be conjugated to a therapeutic agent or drug moiety that modifies a given biological response. The therapeutic agent or drug moiety should not be construed as limited to classical chemotherapeutic agents. For example, the drug moiety can be a protein or polypeptide having the desired biological activity. Examples of such proteins include toxins, cytosines, or growth factors.

  Furthermore, the IgG Fc variant domain containing polypeptide may be conjugated to a therapeutic moiety such as a radioactive substance or macrocyclic chelator useful for binding radioactive metal ions (see above for examples of radioactive metals). it can.

  An antibody comprising a variant IgG Fc domain described herein, ie, an antibody variant, can be conjugated to a therapeutic moiety. Techniques for coupling therapeutic moieties to antibodies are known, see, eg, the following literature: Arnon et al. , “Monoclonal Antibodies for Immunization of Drugs in Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (Eds.), Pp. 243-56. (Alan R. Liss, Inc. 1985); Hellstrom et al. "Antibodies For Drug Delivery", In Controlled Drug Delivery (2nd Ed.), Robinson et al. (Eds.), Pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers of Cytotoxic Agents in Cancer Therapeutics: A Review", in Monoclonal Antibiology. (Eds.), Pp. 475-506 (1985); “Analysis, Results, And Future Prospective of The Therapeutic Use of Radiolabeled Anti-Candocal Therapeutics”, in Monocandoid. (Eds.), Pp. 303-16 (Academic Press 1985), and Thorpe et al. Immunol. Rev. 62: 119-58 (1982). Alternatively, antibody heteroconjugates can be formed by conjugating antibody variants to a second antibody, as described by Segal in US Pat. No. 4,676,980.

  In certain embodiments, an IgG Fc variant domain-containing polypeptide comprises one or more modified glycoforms, ie, a carbohydrate composition that is covalently linked to the polypeptide. Modified glycoforms can be useful for a variety of purposes such as, but not limited to, reducing effector function. The modified glycoform can be obtained by any method known to those skilled in the art, for example, using a modified or mutated expression strain, one or more enzymes (for example, DI N-acetylglucosaminyltransferase III (GnTI11)) and Co-expression, expression of IgG Fc variant domain-containing polypeptides in different organisms or cell lines derived from different organisms, or modification of carbohydrates after expression of IgG Fc variant domain-containing polypeptides it can. Methods for making modified glycoforms are known in the art and include, but are not limited to, those described in the following literature: Umana et al. Nat. Biotechnol. 17: 176-180 (1999); Davies et al. Biotechnol. Bioeng. 74: 288-294 (2001); Shields et al. , J .; Biol. Chem. 277: 26733-26740 (2002); Shinkawa et al. , J .; Biol. Chem. 278: 3466-3473 (2003); Okazaki et al. , J .; Mol. Biol. 336: 1239-49 (2004); US Pat. No. 6,602,684; US 2009/0004179; WO 00/61739, WO 01/292246, International Publication No. 02/311140, International Publication No. 02/30954, International Publication No. 07/005786.

Fusion Protein An Fc fusion protein binds an Fc domain of an immunoglobulin or fragment thereof and a fusion partner, generally any protein, polypeptide, peptide, or small molecule. The role of the non-Fc portion of the Fc fusion protein, ie, the fusion partner, is not necessarily, but is often functionally similar to the variable region of an antibody because it mediates binding to the target. . Accordingly, a fusion partner is provided that specifically binds to a fusion protein, ie, an IgG Fc variant domain-containing polypeptide, and a molecule (eg, a cell surface receptor, chemokine, etc.).

  In certain embodiments, the fusion protein may comprise an antibody mimetic fused to a peptide, polypeptide, protein scaffold, scFv, dsFv, diabody, Tandab (tandem diabody), or IgG Fc variant domain containing polypeptide. . In certain embodiments, the fusion protein may comprise a linker region that connects the peptide, polypeptide, protein scaffold, scFv, dsFv, diabody, Tandab, or antibody mimetic and IgG Fc variant domain-containing polypeptide. The use of natural and artificial peptide linkers to join polypeptides into new linked fusion polypeptides is well known from the literature (Hallewell et al., J. Biol. Chem. 264, 5260-). 5268 (1989); Alfthan et al., Protein Eng. 8, 725-731 (1995); Robinson & Sauer, Biochemistry 35, 109-116 (1996); -32197 (1997); Fares et al. (1998), Endocrinology 139, 2459-2464; Smallshaw et al. (1999), Protein Eng. 2,623-630; U.S. Pat. No. 5,856,456).

  In certain embodiments, the fusion protein may be one that binds a variant IgG Fc domain to a fusion partner, and the fusion partner may generally be a protein, such as but not limited to a ligand, Enzymes, ligand portions of receptors, adhesion proteins, or any other protein or domain. For example, Chamow et al. , Trends Biotechnol. 14: 52-60 (1996); Ashkenazi et al. Curr. Opin. Immunol. 9: 195-200 (1997); Heidaran et al. , FASEB J. et al. 9: 140-5 (1995).

  In one aspect, the fusion protein may comprise a variant IgG Fc domain fused to a moiety that specifically binds to the target molecule, where the target molecule is, for example, a ligand, receptor or fragment thereof. The fusion protein may comprise a variant IgG Fc domain comprising an amino acid substitution as described above, eg, FQQ, FAQ, or FQG Fc domain mutation, one selected from the group consisting of EU positions 252, 254, and 256 Or it may further comprise substitutions at multiple positions, eg, Y, YT, YE, YTE Fc domain mutations.

  In a specific embodiment, the fusion protein comprises a variant IgG Fc domain comprising phenylalanine (F) at EU position 234, glutamine (Q) at EU position 235, and glutamine (Q) at EU position 322. In certain embodiments, the fusion protein comprising an FQQ variant IgG Fc domain further comprises tyrosine (Y) at EU position 252, threonine (T) at EU position 254, and glutamic acid (E) at EU position 256.

  In another embodiment, the fusion protein comprises a variant IgG Fc domain comprising phenylalanine (F) at EU position 234, glutamine (Q) at EU position 235, and glycine (G) at EU position 331. In some embodiments, the fusion protein comprising an FQG variant IgG Fc domain further comprises tyrosine (Y) at EU position 252, threonine (T) at EU position 254, and glutamic acid (E) at EU position 256.

  In yet another aspect, the fusion protein comprises a variant IgG Fc domain comprising phenylalanine (F) at EU position 234, alanine (A) at EU position 235, and glutamine (Q) at EU position 322. In certain embodiments, the fusion protein comprising the FAQ variant IgG Fc domain further comprises tyrosine (Y) at EU position 252, threonine (T) at EU position 254, and glutamic acid (E) at EU position 256.

  In another embodiment, the fusion protein comprises a bioactive molecule fused to a variant IgG Fc domain described herein. Bioactive molecules that can be fused to the variant IgG Fc domains described herein include, but are not limited to, peptides, polypeptides, proteins, small molecules, mimetics, synthetic drugs, inorganic molecules, and Organic molecules are mentioned. In one aspect, the bioactive molecule is at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least A polypeptide comprising 100 contiguous amino acid residues, which is heterologous to the amino acid sequence of the variant IgG Fc domain described herein.

  A fusion protein comprising a variant IgG Fc domain described herein can be fused to a marker sequence such as, but not limited to, a peptide to facilitate purification. In some embodiments, the marker amino acid sequence is one of a His6 tag, a “flag” tag, a hemagglutinin “HA” tag, or various other commercially available tags.

  Fusion proteins can be made by covalently attaching an IgG Fc variant domain-containing polypeptide to a fusion partner using a variety of linkers. Alternatively, polypeptides, proteins and fusion proteins can be produced by standard recombinant DNA techniques, or protein synthesis techniques, eg, peptide synthesizers.

Recombinant polypeptide expression Recombinant expression of an IgG Fc variant domain-containing polypeptide, derivative, analog or fragment thereof, eg, an antibody variant or fusion protein comprising a variant IgG Fc domain as described herein, can This can be achieved by construction of an expression vector comprising a polynucleotide encoding the peptide. Once a polynucleotide encoding an IgG Fc variant domain-containing polypeptide (eg, antibody variant or fusion protein) is obtained, a vector for polypeptide production can be produced by recombinant DNA techniques known in the art. it can.

  Accordingly, methods for making a protein by expressing a polynucleotide comprising a nucleotide sequence encoding an IgG Fc variant domain-containing polypeptide (eg, antibody variant or fusion protein) are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Accordingly, a replicable vector comprising a nucleotide sequence encoding an IgG Fc variant domain containing polypeptide operably linked to a promoter is provided.

  An IgG Fc variant domain-containing polypeptide can be produced by transferring an expression vector to a host cell by conventional techniques and then culturing the transfected cells by conventional techniques. Accordingly, a host cell comprising a polynucleotide encoding an IgG Fc variant domain containing polypeptide operably linked to a heterologous promoter is provided.

  A variety of host expression vector systems can be used to express the IgG Fc variant domain-containing polypeptide (see, eg, US Pat. No. 5,807,715). Such a host expression system represents the vehicle used to generate the coding sequence of interest and subsequently purify it, but when transformed or transfected with an appropriate nucleotide coat sequence, a mutant IgG Fc domain is obtained. Also represented are cells capable of expressing the containing polypeptide in situ. Such host expression systems include, but are not limited to: recombinant bacteriophage DNA, plasmid DNA or one containing one or more sequences encoding an IgG Fc variant domain-containing polypeptide Microorganisms such as bacteria transformed with cosmid DNA (eg, E. coli and B. subtilis); a set comprising one or more sequences encoding an IgG Fc variant domain-containing polypeptide Yeast transformed with a recombinant yeast expression vector (eg, Saccharomyces Pichia); a recombinant viral expression vector (eg, baculovirus) comprising one or more sequences encoding an IgG Fc variant domain-containing polypeptide Virus) Insect cell line; infected with a recombinant viral expression vector (eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) comprising one or more sequences encoding an IgG Fc variant domain containing polypeptide or IgG A plant cell line transformed with a recombinant plasmid expression vector (eg, Ti plasmid) comprising one or more sequences encoding an Fc variant domain-containing polypeptide; or alternatively derived from the genome of a mammalian cell or a mammalian virus A mammalian cell line carrying a recombinant expression construct containing the promoter of (eg, COS, CHO, BHK, 293, NS0, 3T3 cells).

  A host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (eg, glycosylation) and processing (eg, cleavage) of protein products can be important for the function of the protein. Different host cells have unique and unique mechanisms for the post-translational processing and modification of proteins and gene products. Eukaryotic host cells with cellular mechanisms for proper processing of the primary transcript, glycosylation of the gene product, and phosphorylation can be used. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, HeLa, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, NS0, CRL7O3O and HsS78Bst. Cell.

  For long-term, high yield production of recombinant proteins, stable expression is often preferred. For example, a cell line that stably expresses an IgG Fc variant domain-containing polypeptide can be produced using methods known in the art.

  Once the IgG Fc variant domain-containing polypeptide (eg, antibody variant or fusion protein) is produced by recombinant expression, protein purification methods known in the art, eg, chromatography (eg, ion exchange chromatography, particularly Protein A can be purified by affinity chromatography with affinity for a specific antigen followed by sizing column chromatography), centrifugation, differential solubility, or any other standard protein purification method.

Characterization and Functional Assays IgG Fc variant domain containing polypeptides can be characterized in a variety of ways. In particular, IgG Fc variant domain-containing polypeptides can be assayed for the ability to specifically bind to a ligand, such as FcγRIIA, FcγRIIIA (158V), Clq. Such assays are performed in solution (see, for example, Houghten, Bio / Techniques 13: 412-421 (1992)), using beads (see, for example, Lam, Nature 354: 82-84). (See, eg, Fodor, Nature 364: 555-556 (1993)), using bacteria (see, eg, US Pat. No. 5,223,409), and using plasmids (see, eg, Cull et al. al., Proc. Natl. Acad. Sci. USA 89: 1865-1869 (1992)) or using phage (eg, Scott and Smith, Science 249: 386-390 (1990); Devlin, Science 249. : 404-406 (199 Cirla et al., Proc. Natl. Acad. Sci. USA 87: 6378-6382 (1990); and Felici, J. Mol. Biol. 222: 301-310 (1991)). it can. Molecules found to bind specifically to ligands such as FcγRIIA, FcγRIIIA (158V), Clq can then be assayed for their affinity for the ligand.

  IgG Fc variant domain-containing polypeptides can be specifically bound to molecules such as antigens (eg, cross-reactive with cancer antigens and other antigens) or ligands (eg, FcγR) by any method known in the art. Can be assayed for. Immunoassays that can be used to analyze specific binding and cross-reactivity include, but are not limited to, competitive and non-competitive assay systems using techniques such as Western blot, radioimmunoassay, ELISA (enzyme binding) Immunosorbent assays), “sandwich” immunoassays, immunoprecipitation assays, sedimentation reactions, agglutination assays, complement fixation assays, fluorescent immunoassays, protein A immunoassays, and the like. Such assays are routine and well known in the art. For example, Ausubel et al. , Eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc. , New York.

  The binding affinity of an IgG Fc variant domain-containing polypeptide for a molecule such as an antigen or ligand (eg, FcγR) and the rate of dissociation of the interaction can be determined in a competitive binding assay. The kinetic parameters of IgG Fc variant domain-containing polypeptides can also be determined using any surface plasmon resonance (SPR) based assay known in the art (eg, BIAcore or ProteOn kinetic analysis). See, for example, Mullet et al. Methods 22: 77-91 (2000); Dong et al. Rev. Mol. Biotech. 82: 303-23 (2002); Fivash et al. Curr. Opin. Biotechnol. 9: 97-101 (1998); Rich et al. Curr. Opin. Biotechnol. 11: 54-61 (2000). In addition, U.S. Patent No. 6,373,577; U.S. Patent No. 6,289,286; U.S. Patent No. 5,322,798; U.S. Patent No. 5,341,215; Any of the SPR instruments and SPR-based methods for measuring protein-protein interactions described in US Pat. No. 6,268,125 are contemplated in the disclosed method.

  Fluorescence activated cell sorting using any of the techniques known to those skilled in the art to characterize the binding of an IgG Fc variant domain containing polypeptide to a cell surface expressed molecule (eg, FcγR) Act (FACS) can be used.

  An IgG Fc variant domain-containing polypeptide can be assayed for its ability to mediate FcγR-mediated effector cell function. Examples of effector cell functions that can be assayed include, but are not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), C1q binding, and complement-dependent cell-mediated cytotoxicity (CDC). . Any cell-based or cell-free assay known to those skilled in the art can be used to determine effector cell functional activity (eg, Perusia et al. Methods Mol. Biol. 121: 179-92 (2000)). Baggiolini et al.Experientia 44: 841-8 (1998); Lehmann et al. J. Immunol.Methods 243: 229-42 (2000); Brown, Methods Cell Biol.45: 147-un (1994); al.J.Exp.Med.172: 231-237 (1990); Abdul-Majid et al., Scand. J. Immunol.55: 70-81 (2002); et al.Immunity 8: 403-411 (1998)). In particular, IgG Fc variant domain-containing polypeptides can be assayed for FcγR-mediated ADCC activity in effector cells such as natural killer cells using any of the standard methods known to those skilled in the art (eg, Perusia). et al., Methods MoI.Biol.121: 179-92 (2000)).

  Methods for characterizing the ability of IgG Fc variant domain containing polypeptides to bind to C1q and mediate complement dependent cytotoxicity (CDC) are known in the art. For example, to determine C1q binding, a C1q binding ELISA can be performed. To assay complement activation, see, for example, Gazzano-Santoro et al. J. et al. Immunol. Complement dependent cell (CDC) assays can be performed as described in Methods 202: 163 (1996).

Pharmaceutical compositions and methods of administration In another aspect, a composition comprising an IgG Fc variant domain-containing polypeptide formulated with a carrier, a nucleic acid encoding an IgG Fc variant domain-containing polypeptide, or a combination thereof. Provided. Such compositions can include one antibody or a combination of (eg, two or more different) antibodies, a fusion protein, or a conjugate. In certain embodiments, such compositions are physiologically tolerated and are therefore suitable for therapeutic, prophylactic, or diagnostic administration to a subject.

  In another embodiment, the composition comprising an IgG Fc variant domain-containing polypeptide (eg, antibody variant, fusion protein, or conjugate) or a nucleic acid encoding an IgG Fc variant domain-containing polypeptide is one or more. Species pharmaceutically acceptable salts may be included.

  Examples of suitable aqueous and non-aqueous carriers that can be used in the contemplated composition include water, ethanol, polyols (eg, glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, such as olive oil Examples include organic esters for injection such as vegetable oil and ethyl oleate. For example, proper fluidity can be maintained by using a coating material such as lecithin, maintaining the required particle size in the case of a dispersion, and using a surfactant.

  In another aspect, a composition comprising an IgG Fc variant domain-containing polypeptide (eg, antibody variant, fusion protein, or conjugate) or a nucleic acid encoding an IgG Fc variant domain-containing polypeptide further comprises a preservative. And agents such as wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms can be ensured both by sterilization methods and the inclusion of various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol sorbic acid and the like. It may also be desirable to include isotonic agents such as sugars and sodium chloride in the composition. Moreover, long-term absorption of injectable pharmaceutical forms can be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

  Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Unless conventional media or agents are incompatible with the active compounds, their use in pharmaceutical compositions is contemplated. Additional active compounds can also be incorporated into the compositions. In certain embodiments, acceptable carriers include excipients that are approved for human or animal administration or deemed safe for such administration, ie, GRAS substances (generally considered safe). GRAS substances are described by the US Food and Drug Administration in Code of Federal Regulations (CFR) 21 CFR 182 and 22 CFR 182 which are hereby incorporated by reference. Shall be incorporated into

  The actual amount of active ingredient used in a pharmaceutical composition comprising an IgG Fc variant domain-containing polypeptide (eg, antibody variant, fusion protein or conjugate) or a nucleic acid encoding an IgG Fc variant domain-containing polypeptide is The therapeutic response desired for a particular patient, composition, and method of administration can be varied to obtain an amount of active ingredient effective to achieve the patient's toxicity without becoming toxic. The amount used is used in combination with the specific composition used, or its ester, salt or amide, route of administration, administration time, elimination rate of the specific compound used, duration of treatment, specific composition used. Various pharmacokinetics, including other drugs, compounds and / or substances, age, gender, weight, medical condition, general health status and previous medical history of the patient to be treated, and similar factors known in the medical field It depends on the factors.

  A therapeutically effective dose of an IgG Fc variant domain containing polypeptide, a nucleic acid encoding an IgG Fc variant domain containing polypeptide, or a pharmaceutical composition thereof reduces the severity of disease symptoms, the frequency of asymptomatic periods and Increased length or prevention of dysfunction or disability due to morbidity results. A therapeutically effective dose can also prevent or delay disease onset. Thus, clinical or biochemical monitoring assays can be used to determine whether a particular treatment is a therapeutically effective dose. One of ordinary skill in the art can determine such amounts based on factors such as the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration selected.

  Compositions comprising a nucleic acid encoding an IgG Fc variant domain-containing polypeptide (eg, antibody variant, fusion protein, or conjugate) or IgG Fc variant domain-containing polypeptide can be obtained in a variety of ways known in the art. One or more can be used to administer by one or more routes of administration. As will be appreciated by those skilled in the art, the route and / or method of administration will vary depending on the desired result. Routes of administration selected for IgG Fc variant domain-containing polypeptides, compositions comprising nucleic acids encoding IgG Fc variant domain-containing polypeptides, and pharmaceutical compositions thereof include, for example, intravenous or muscle by injection or infusion There are internal, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration. Parenteral administration is an administration method other than enteral or topical administration, and is usually performed by injection, but is not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac Intradermal, intraperitoneal, intratracheal, subcutaneous, epidermal, intraarticular, subcapsular, subarachnoid, intrathecal, epidural and intrasternal injection and infusion. Alternatively, an IgG Fc variant domain-containing polypeptide, a composition comprising a nucleic acid encoding an IgG Fc variant domain-containing polypeptide, and a pharmaceutical composition thereof can be applied to a non-parenteral route such as a topical, epidermal or mucosal route of administration. For example, intranasal, oral, vaginal, rectal, sublingual or topical administration.

Methods of Treatment An IgG Fc variant domain-containing polypeptide (eg, antibody variant, fusion protein, or conjugate) or a nucleic acid encoding an IgG Fc variant domain-containing polypeptide is one associated with a disease, disorder, or infection. Or it can be administered to animals, particularly mammals, specifically humans, to prevent, treat or ameliorate multiple symptoms.

  An IgG Fc variant domain-containing polypeptide or a nucleic acid encoding an IgG Fc variant domain-containing polypeptide is used to treat or prevent a disease or disorder in which the effect of effector cell function (eg, ADCC and / or CDC) is desired. Can be particularly useful.

  IgG Fc variant domain-containing polypeptide or nucleic acid encoding IgG Fc variant domain-containing polypeptide and compositions thereof for the treatment of primary or metastatic tumor disease (ie, cancer), autoimmune diseases, and infectious diseases Or it may be particularly useful for prevention. An IgG Fc variant domain-containing polypeptide or a nucleic acid encoding an IgG Fc variant domain-containing polypeptide can be provided as a pharmaceutically acceptable composition known in the art or as described herein. . As detailed below, an IgG Fc variant domain-containing polypeptide or a nucleic acid encoding an IgG Fc variant domain-containing polypeptide is a cancer (especially in passive immunotherapy), autoimmune disease, inflammatory disease or infection. It can be used in a method for treating or preventing symptom.

  Furthermore, an IgG Fc variant domain-containing polypeptide or a nucleic acid encoding an IgG Fc variant domain-containing polypeptide, and a composition thereof, are used for the treatment or prevention of cancer, autoimmune diseases, inflammatory diseases or infectious diseases. It can also be advantageously used in combination with other therapeutic agents known in the art. An IgG Fc variant domain-containing polypeptide or a nucleic acid encoding an IgG Fc variant domain-containing polypeptide, and compositions thereof can also be used to treat one or more drugs used to treat a disease, disorder, or infection, For example, it can be advantageously used in combination with an anticancer drug, an anti-inflammatory drug or an antiviral drug.

  In certain embodiments, one or more symptoms associated with cancer and related conditions are prevented, treated, or administered by administering an IgG Fc variant domain-containing polypeptide or a nucleic acid encoding an IgG Fc variant domain-containing polypeptide, or A method is provided for improvement.

  An IgG Fc variant domain-containing polypeptide or a nucleic acid encoding an IgG Fc variant domain-containing polypeptide can be used to prevent, treat, or manage one or more symptoms associated with an inflammatory disease in a subject it can.

  The present disclosure also encompasses a method of treating or preventing an infection in a subject comprising administering a therapeutically or prophylactically effective amount of an IgG Fc variant domain-containing polypeptide. Infectious diseases that can be treated or prevented by IgG Fc variant domain-containing polypeptides are caused by infectious agents such as, but not limited to, viruses, bacteria, fungi, protozoa, and viruses.

Kits Further provided are pharmaceutical packs or kits comprising one or more containers filled with one or more pharmaceutical compositions disclosed herein. Optionally, such a container may be provided with a notice in a form prescribed by a government agency that regulates the manufacture, use or sale of the drug or biologic, which notice is for manufacture for human administration. , Representing use or approval by a sales organization. The present disclosure provides kits that can be used in treatment and administration methods. In one aspect, the kit comprises the IgG Fc variant domain-containing polypeptide (eg, antibody variant, fusion protein, or conjugate), preferably in purified form, in one or more containers.

  These examples are described for illustrative purposes only, and are not to be construed as limiting the scope of the claims in any way, but rather include any modifications that will become apparent as a result of the teachings described herein. Should be interpreted.

Example 1
Production and Purification of Mutant IgG Fc Domain and Antibody Generation and Purification Anti-RSV Glycoprotein F Humanized Monoclonal Antibody Motabizumab (MEDI 532, Numax ™; Wu et al., J. Mol. Biol. 350: 126-144 (2005 Wu et al., J. Mol. Biol.368: 652-65 (2007)) (hereinafter referred to as “Ab1”), anti-IL-4R antibody (eg, US Pat. No. 8,092,804). (Refer to the specification) (hereinafter referred to as “Ab2”), anti-CDR20 antibody HB20.3 (see, for example, US 2009/0136516: US 2009/0155275) (hereinafter “ (Referred to as “Ab3”) or the heavy chain Fc region of anti-C5a antibody 1B8 (hereinafter referred to as “Ab4”) did. Mutations were introduced by site-directed mutagenesis using PCR with overlap extension (see, eg, Ho et al., Gene 77: 51-59 (1989)). A region of the heavy chain gene was amplified using primers containing the desired mutation. These fragments were then combined (if necessary) to produce a full-length human IgG1 Fc gene fragment containing the desired mutation. The final PCR fragment was then individually cloned into a heavy chain encoding mammalian expression vector. By this treatment, the wild type heavy chain constant part of the antibody was replaced with a different Fc-modified corresponding part. DNA sequencing used to confirm the construct was performed by Genewiz (South Plainfield, NJ).

  All constructs were transiently expressed in HEK293F cells using 293fectin ™ (Invitrogen) as a transfection reagent and grown in Invitrogen's serum-free Freestyle ™ medium. Medium was collected 10 days after transfection and all antibody formats were purified by standard protein A affinity chromatography according to the manufacturer's protocol (GE Healthcare, Piscataway, NJ). Subsequently, the antibody was buffer exchanged with 25 mM histidine-HCl (pH 6.0), and the purity of the construct was analyzed using SDS-PAGE under reducing and non-reducing conditions, and further by analytical size exclusion chromatography. 99-100% pure IgG samples were obtained using preparative size exclusion chromatography.

Example 2
Differential Scanning Calorimetry (DSC), and SYPRO® Orange Differential Scanning Fluorimetry (DSF) Thermal Stability Measurement IgG domain instability is thought to correlate with unfavorable chemical, manufacturing, and control (CMC) properties Such properties include thermal stability and reduced solubility, increased aggregation or fragmentation, which ultimately increases purity loss, limited formulation / delivery options, and other developmental possibilities. It will cause challenges.

  DSC experiments were performed using a Microcal VP-DSC differential scanning microcalorimeter (Microcal, Northampton, Mass.). All solutions and samples used for DSC were filtered using a 0.22 μm filter and vented before loading into the calorimeter. The antibody used in the DSC test was> 95% monomer as judged by analytical gel filtration chromatography. Prior to DSC analysis, all samples were dialyzed carefully (at least 3 buffer exchanges) with 25 mM histidine-HCl (pH 6). The buffer from this dialysis was used as a reference buffer for subsequent DSC experiments. Prior to sample measurement, baseline measurements (buffer vs. buffer) were obtained and subtracted from the sample measurements. Dialyzed sample (concentration 1 mg / ml) was added to the sample well and DSC measurements were performed at a scan rate of 1 ° C./min. Data analysis and deconvolution were performed using Origin ™ DSC software provided by Microcal.

For DSF experiments, SYPRO® Orange was added to the antibody at a concentration of 0.5 mg / ml in 25 mM histidine-HCl (pH 6) (Goldberg et al., J. Pharm. Sci. 100: 1306). -1315 (2011)). 25 microliters of the prepared sample was added twice to the white PCR plate. Chromo4 Real Time PCR Detector (Bio-Rad, Hercules, Calif.) Was used as a thermal cycler and fluorescence emission was detected using a software custom dye calibration routine. The PCR plate containing the test sample was subjected to a temperature increase from 20 ° C. to 90 ° C. in 0.2 ° C. increments, with a 10 second interval between each temperature increment. _Tm was calculated by software using a mathematical quadratic function method to calculate the inflection point of the curve. The recorded T _m is the average of the three measurements.

  Mutations were introduced into the Fc region of Ab1 or Ab2. FES and YTE mutations were introduced into the Fc domains of Ab1 and Ab2 (see data corresponding to IgG1-FES-YTE in Table 4), and decreased thermal stability and increased purity loss were observed. Ab1 and Ab2 showed 14 ° C and 13 ° C losses in thermal stability, respectively (measured by DSC). The FES-YTE antibody variant (IgG1-FES-YTE in Table 5) also showed significantly increased purity loss relative to either the wild type or YTE counterpart (using an accelerated stability assay, 1 Measured at 40 ° C. for months).

Thermal stability upon mutation of the YTE set or addition of FES-YTE only affected the CH2 domain (FIG. 1). From the DSC trace of Ab4, it was found that the CH2 domain T _m is reduced by the addition of YTE and FES-YTE mutations, while the thermal stability of the Fab and CH3 domains is maintained unaffected. Although the addition of YTE reduced thermostability (see Table 4 and FIG. 1), a significant increase in purity loss began only when the FES mutation was introduced in combination with the YTE mutation.

  In order to assist in the construction of higher thermostable mutant Fc domains with the same biological properties as mutant Fc domains containing FES-YTE mutations, we The contribution of FES mutations to thermostability was determined and the results showed that EU L234F mutation had no effect on thermostability whereas EU L235E and EU P331S mutations were -Decreased thermal stability of about -2.5 ° C and -3.5 ° C, respectively, as determined by DSC, relative to YTE background (ie, the Ab3 antibody whose Fc domain already contains a YTE mutation) EU L234F, EU L235E, and EU P331S mutations were introduced).

  Another mutation was made at the L235 and P331 EU positions of the Fc domain and evaluated for increased thermal stability (Table 6). Since the EU P331S substitution was first included in the FES mutant with the aim of reducing C1q binding, we also made another site, EU position K322 (also C1q Selected to reduce binding) (Idusogie et al., J. Immunol. 164: 4178-4184 (2000)).

  All antibodies containing variant Fc domains with selected mutations (I, A, N, F, Q and V) at EU position L235 are compared with corresponding antibodies containing variant Fc domains with EU L235E mutation Exhibited an increased thermal stability of about 2 ° C. in the L234F YTE Ab3 background.

  At EU position P331, the alanine and glycine mutations improved thermostability only slightly (1 ° C.) compared to antibodies containing Fc domains with FES-YTE mutations (mutations were EU L234F). L235E YTE Ab3 background). Mutations to A, E, N, H and Q were made at EU position K322 (an alternative site to EU position P331) and analyzed for increased thermal stability. EU K322E, K322N and K322H substitutions actually resulted in reduced thermal stability compared to FES-YTE. When these mutations were introduced into the EU L234F L235E YTE Ab3 background, CH2 Tm decreased by 0.3, 7, and 2.6 ° C., respectively. EU K322A and K322Q mutations to the EU L234F L235E YTE Ab3 background resulted in improved thermal stability of 1.2 ° C and 2.8 ° C, respectively, compared to FES-YTE.

  The EU is then evaluated to assess improved thermal stability, purity loss and biophysical stability, and desired biological properties such as increased FcRn binding and lack of ADCC and CDC induction. Constructs were generated that combined the most thermostable mutations at positions L235 and P331 or K322.

  FQG-YTE, FQQ-YTE, and FAQ-YTE mutant Fc domains were formed in the Ab3 and Ab4 backgrounds. Ab3 and Ab4 mutants containing FQG-YTE, FQQ-YTE, and FAQ-YTE mutant Fc domains are significantly more thermally stable compared to Ab3 and Ab4 mutants containing the FES-YTE mutation (FIG. 1). The improvement of property was shown (FIG. 2).

In Ab3 background, FES-YTE mutations had a CH2 _{T m} of 55.6 ° C.. In contrast, Ab3 mutants containing the FAQ-YTE, FQG-YTE, and FQQ-YTE mutations have CH2 T _m of 60.6 ° C., 60.2 ° C., and 61.6 ° C., respectively (Table 7) This indicates that these combinations of Fc domain mutations actually conferred improved thermal stability to the CH2 domain.

Example 3
Surface Plasmon Resonance Binding Analysis An antibody comprising a combination of three mutants FQQ, FQF, and FAQ, and a variant IgG Fc domain with a YTE mutation, which is a variant IgG Fc domain with a FES-YTE mutation Were tested for binding to the FcγR receptor, C1q and FcRn.

  Experiments were performed using a ProteOn XPR36 surface plasmon resonance apparatus (Bio Rad). Phosphate buffered saline containing 0.005% Tween 20 (PBS / Tween), pH 7.4 was used as the running buffer for most experiments except for human FcRn (extracellular domain) binding. For human FcRn binding, phosphate buffered saline containing 0.005% Tween 20 (PBS / Tween), pH 6.0 was used. All experiments were performed at 25 ° C. Using EDAC (1-ethyl-3- [3-dimethylaminopropyl] carbodiimide) and sulfo-NHS (N-hydroxysulfosuccinimide) on a ProteoOn ™ GLC Sensor Chip (# 176-5011) 6000 R.D. U. The antibody was immobilized to the (response unit) level. Next, C1q, FcγRI, FcγRIIa, FcγRIIb, FcγRIII (158V), FcγRIII (158F), and FcRn at various concentrations on the IgG-immobilized surface at a flow rate of 25 μl / min, or almost steady state For enough time to achieve. Binding (if any) can be quantified by steady state equilibrium binding analysis using ProteOn software. Human FcγRI, FcγRIIa, FcγRIIb, FcγRIII (158V), FcγRIII (158F), and FcRn extracellular domain were generated in situ with mammalian expression vectors. Human C1q was obtained from Quidel (CA).

  An antibody comprising a variant IgG Fc domain having a FES-YTE, FAQ-YTE, FQG-YTE, or FQQ-YTE mutation is FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa (158V), FcγRIIIa (158F), or C1q. It did not show quantifiable binding by SPR (ProteOn) (Tables 8 and 9). FcRn binding to an antibody comprising a variant IgG Fc domain having a FQQ-YTE or FQG-YTE mutation was similar to the value obtained for an antibody comprising a variant IgG Fc domain having a FES-YTE or YTE mutation. However, this indicates that improved FcRn binding is maintained at pH 6.0 (Tables 8 and 9).

  Thus, from SPR binding experiments to cell receptors known to be essential for ADCC for antibodies containing variant IgG Fc domains with FAQ-YTE, FQG-YTE, or FQQ-YTE mutations And the soluble receptor C1q that can initiate CDC was found to be lacking.

Example 4
Antibody Dependent Cytotoxicity (ADCC) Assay An antibody dependent cellular cytotoxicity (ADCC) assay was performed using a variant IgG Fc domain incorporated into the Ab3 (HB20.3 anti-CD20; SEQ ID NOs: 5 and 6) background. Natural killer cell lines expressing CD16 were used as effector cells and Daudi cells as targets. Daudi cells and effector cells were washed with PBS and diluted to a density of 800,000 c / ml. Daudi cells and effector cells were added to white V-shaped well bottom 96-well plates (50 μl each). Various concentrations of Ab3 antibody variants were added to the wells. Cells and antibodies were incubated for 4 hours at 37 ° C. Daudi cell death was also monitored by analysis of LDH release using the CytoTox 96 non-radioactive cytotoxicity assay (Promega) according to the manufacturer's instructions.

  In an experiment using a natural killer (NK) cell-derived cell line as an effector cell and a Daudi cell as a target, WT Ab3 antibody and Ab3 antibody containing a mutant IgG Fc domain having a YTE mutation can be titrated. Showed a response. Conversely, Ab3 antibodies containing mutant IgG Fc domains with FAQ-YTE, FQQ-YTE, or FES-YTE mutations did not induce significant cytotoxicity even at concentrations up to 300 μg / ml (FIG. 3). ).

Example 5
Complement Dependent Cytotoxicity (CDC) Assay Complement dependent cytotoxicity (CDC) assay was performed using a variant IgG Fc domain incorporated into Ab3 (HB20.3 anti-CD20; SEQ ID NOs: 5 and 6). Human serum from a healthy donor was used as a complement source and Daudi cells were used as effector cells. Daudi cells were washed with PBS and diluted to a density of 800,000 c / ml. The cells were then added to white V-bottom plates (50 μl each). Diluted serum (50 μl) was then added at the desired concentration along with various dilutions of antibody. Cells were incubated for 4 hours at 37 ° C. Subsequently, alamar blue reagent (Life Technologies) was added and after 12 hours Daudi cell death was quantified (according to manufacturer's instructions).

  Only WT Ab3 antibodies or Ab3 antibodies containing Fc domains with YTE mutations were found to induce robust complement dependent cytotoxicity. Conversely, Ab3 antibodies containing mutant IgG Fc domains with FAQ-YTE, FQG-YTE, FQQ-YTE, FES-YTE mutations ended without inducing complement-dependent cytotoxicity (FIG. 4).

Example 6
Accelerated Stability Test Ab4 antibody (1B8 anti-C5a antibody; SEQ ID NOs: 7 and 8) antibody variants in the background were concentrated to 100 mg / ml in 25 mM histidine pH 6.0. The antibody was placed in a glass vial and stored for 6 weeks in a controlled room (40 ° C., 75% relative humidity). Samples were tested weekly by high performance size exclusion chromatography (HPSEC) for percent aggregate. HPSEC analysis was performed on an Agilent HPLC instrument (Agilent Technologies) equipped with a TSK-Gel G3000 column. Eluted protein was detected using UV absorbance at 280 nm and the results were recorded as product monomer peak area percentage (%). The peak eluting earlier than the monomer was recorded as the aggregate percentage (%), and the peak eluting after the monomer was recorded as the fragment / other percentage (%). Aggregation rate over time was determined by linear regression.

  Accelerated stability studies were performed on antibodies containing mutant IgG Fc domains with FAQ-YTE, FQG-YTE, FQQ-YTE mutations versus antibodies containing mutant IgG Fc domains with FES-YTE mutations. The improvement observed in the aggregation / fragmentation rate was evaluated (Table 10). The FES-YTE antibody variant (in the Ab4 background) had a 1.9% aggregation rate at 40 ° C. for 1 month, whereas the Ab4 antibodies with FQG-YTE and FQQ-YTE mutations, respectively, An improvement from 1.01% to 1.1% was shown.

  The Ab4 antibody with the FES-YTE mutation had an overall monomer loss rate of 3.82% per month, whereas the WT Ab4 antibody and the Ab4 antibody with the YTE mutation were each 2.71 per month. % And 2.75% overall monomer loss. Ab4 antibodies with FQQ-YTE and FQG-YTE mutations are 3.28% and 3.36%, an improved overall monomer loss rate at 40 ° C. compared to Ab4 antibodies with FES-YTE mutation Had. The Ab4 antibody with the FAQ-YTE mutation had higher monomer loss / month than expected due to the higher fragmentation rate (5.74%).

Example 7
Isoelectric focusing (IEF) gel A precast amphorine gel (Amersham Biosciences, Uppsala Sweden; pI range 3.5-9.5) was loaded with IgG. Relative pI was determined for various antibodies or antibody fragments using a broad range pI marker standard (Amersham Biosciences, pI range 3-10). Electrophoresis was performed at 1500 V and 50 mA for 105 minutes. The gel was fixed for 45 minutes using Sigma fixative (Sigma, Saint Louis, MO). Staining was performed overnight at room temperature using Simply Blue dye (Invitrogen). Destaining was performed using a solution consisting of 25% ethanol, 8% acetic acid and 67% purified water.

  From analysis by IEF gel, introduction of FAQ-YTE, FQG-YTE, and FQQ-YTE mutations into the Fc domain of Ab4 antibody (1B8 anti-C5a antibody; SEQ ID NOs: 7 and 8) is minimal for pI All the constructs were found to have an approximate pI> 8.3, affecting only (FIG. 5).

Example 8
Apparent Solubility by Polyethylene Glycol (PEG) Precipitation The PEG precipitation assay is described in Gibson et al. , J .; Pharm. Sci. 100: 1009-1021 (2011). The antibody was precipitated by adding increasing amounts of PEG 6000. PEG 6000 solutions ranging from 0% to 40% [w / v] PEG in 50 mM sodium phosphate buffer pH 7.2 were prepared. These solutions were added to the wells of a 96 well white polystyrene filter plate. The antibody was then added to wells containing various levels of PEG to a final concentration of 1 mg / mL. The 96 well plate was incubated overnight at room temperature. The plate was then centrifuged and the filtrate was collected in a clear polystyrene 96 well collection plate. Subsequently, an equal amount of each sample filtrate was transferred to a new transparent polystyrene 96-well plate, and the filtrate was analyzed for protein concentration by measuring the absorbance at 280 nm using a nanodrop. Apparent solubility is calculated using the slope of the aggregation transition.

  Ab4 antibodies (1B8 anti-C5a antibodies; SEQ ID NOs: 7 and 8) containing mutant Fc domains with FAQ-YTE, FQG-YTE, and FQQ-YTE mutations were evaluated for improved apparent solubility, Evaluation was by precipitation (Table 11). A measure of “apparent solubility” can be obtained by extrapolation of agglomerated transitions. This value is useful only for grading antibody variants because absolute values do not represent actual solubility limits (eg, values for highly soluble proteins are overestimated).

  The Ab4 antibody containing a variant Fc domain with the FES-YTE mutation showed the lowest apparent solubility of all antibodies tested at <10 mg / ml. All Ab4 antibodies containing mutant Fc domains with FAQ-YTE, FQG-YTE, FQQ-YTE had a high apparent solubility score> 100 mg / ml and showed improved solubility for FES-YTE.

Example 9
Dynamic light scattering (DLS)
Protein particle size distribution and molecular size were monitored by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern Instruments, Malvern, PA). A sample (1 mg / ml in 25 mM histidine-HCl pH 6) was irradiated using a 633 nm laser, and the intensity of the scattered light was measured at an angle of 173 °. Samples analyzed at room temperature were incubated for approximately 90 minutes on a laboratory bench top and then sampled. Prior to each sample analysis, correction factors were introduced for parameters such as viscosity, refractive index and absorbance.

  Variants of Ab4 antibodies (1B8 anti-C5a antibodies; SEQ ID NOs: 7 and 8) containing FAQ-YTE, FQG-YTE, FQQ-YTE mutations in their respective Fc domains, and FES- in wild-type Ab4 and their respective Fc domains All variants containing YTE and YTE mutations were found to be monodisperse by dynamic light scattering (DLS) (Table 12). Therefore, even when each combination of the FAQ-YTE, FQG-YTE, and FQQ-YTE mutations was introduced into the Fc domain of the Ab4 antibody, no increase in polydispersity occurred.

  From the above description of specific embodiments, others can apply the basic knowledge in the field to facilitate the above specific embodiments without undue experimentation without departing from the overall concept described. The overall nature of the present disclosure will become fully apparent as it can be changed and / or modified for various applications. Accordingly, such modifications and changes are intended to be included within the meaning and scope of the equivalents of the disclosed aspects based on the teachings and guidelines presented herein. The terms and expressions herein are for illustrative purposes and are not intended to be limiting, as the terms and expressions herein are interpreted by one of ordinary skill in the art in light of the present teachings and guidelines. It should be understood that there is no.

  The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

[１] 変異型ＩｇＧ Ｆｃドメインを含む単離されたポリペプチドであって、前記変異型ＩｇＧ Ｆｃドメインが、
（ａ）２３４位にフェニルアラニン（Ｆ）アミノ酸；
（ｂ）２３５位にアラニン（Ａ）、アスパラギン（Ｎ）、フェニルアラニン（Ｆ）、グルタミン（Ｑ）、若しくはバリン（Ｖ）アミノ酸；及び
（ｃ）３２２位にアラニン（Ａ）、アスパラギン酸（Ｄ）、グルタミン酸（Ｅ）、ヒスチジン（Ｈ）、アスパラギン（Ｎ）、若しくはグルタミン（Ｑ）アミノ酸；又は３３１位にアラニン（Ａ）若しくはグリシン（Ｇ）アミノ酸
を有し、
ここで、前記アミノ酸番号付けは、Ｋａｂａｔに記載のＥＵインデックスに従う、単離されたポリペプチド。
[２] ２３４位にフェニルアラニン（Ｆ）アミノ酸；２３５位にグルタミン（Ｑ）アミノ酸；及び３２２位にグルタミン（Ｑ）アミノ酸を有し、ここで、前記アミノ酸番号付けは、Ｋａｂａｔに記載のＥＵインデックスに従う、１に記載のポリペプチド。
[３] ２３４位にフェニルアラニン（Ｆ）アミノ酸；２３５位にグルタミン（Ｑ）アミノ酸；及び３３１位にグリシン（Ｇ）アミノ酸を有し、ここで、前記アミノ酸番号付けは、Ｋａｂａｔに記載のＥＵインデックスに従う、１に記載のポリペプチド。
[４] ２３４位にフェニルアラニン（Ｆ）アミノ酸；２３５位にアラニン（Ａ）アミノ酸；及び３２２位にグルタミン（Ｑ）アミノ酸を有し、ここで、前記アミノ酸番号付けは、Ｋａｂａｔに記載のＥＵインデックスに従う、１に記載のポリペプチド。
[５] （ａ）２５２位にチロシン（Ｙ）アミノ酸、若しくは２５２位にセリン（Ｓ）アミノ酸、若しくは２５２位にトリプトファン（Ｗ）アミノ酸、若しくは２５２位にトレオニン（Ｔ）アミノ酸；及び／又は
（ｂ）２５４位にトレオニン（Ｔ）アミノ酸；及び／又は
（ｃ）２５６位にグルタミン酸（Ｅ）アミノ酸、若しくは２５６位にセリン（Ｓ）アミノ酸、若しくは２５６位にアルギニン（Ｒ）アミノ酸、若しくは２５６位にグルタミン（Ｑ）アミノ酸、若しくは２５６位にアスパラギン酸（Ｄ）アミノ酸
をさらに有し、
ここで、前記アミノ酸番号付けは、Ｋａｂａｔに記載のＥＵインデックスに従う、１〜４のいずれか１項に記載のポリペプチド。
[６] （ａ）２５２位にチロシン（Ｙ）アミノ酸；及び／又は
（ｂ）２５４位にトレオニン（Ｔ）アミノ酸；及び／又は
（ｃ）２５６位にグルタミン酸（Ｅ）アミノ酸
をさらに有し、
ここで、前記アミノ酸番号付けは、Ｋａｂａｔに記載のＥＵインデックスに従う、１〜４のいずれか１項に記載のポリペプチド。
[７] （ａ）２５２位にチロシン（Ｙ）アミノ酸、若しくは２５２位にセリン（Ｓ）アミノ酸、若しくは２５２位にトリプトファン（Ｗ）アミノ酸、若しくは２５２位にトレオニン（Ｔ）アミノ酸；及び
（ｂ）２５４位にトレオニン（Ｔ）アミノ酸
をさらに有し、
ここで、前記アミノ酸番号付けは、Ｋａｂａｔに記載のＥＵインデックスに従う、１〜４のいずれか１項に記載のポリペプチド。
[８] （ａ）２５４位にトレオニン（Ｔ）アミノ酸；及び
（ｂ）２５６位にグルタミン酸（Ｅ）アミノ酸、若しくは２５６位にセリン（Ｓ）アミノ酸、若しくは２５６位にアルギニン（Ｒ）アミノ酸、若しくは２５６位にグルタミン（Ｑ）アミノ酸、若しくは２５６位にアスパラギン酸（Ｄ）アミノ酸
をさらに有し、
ここで、前記アミノ酸番号付けは、Ｋａｂａｔに記載のＥＵインデックスに従う、１〜４のいずれか１項に記載のポリペプチド。
[９] （ａ）２５２位にチロシン（Ｙ）アミノ酸、若しくは２５２位にセリン（Ｓ）アミノ酸、若しくは２５２位にトリプトファン（Ｗ）アミノ酸、若しくは２５２位にトレオニン（Ｔ）アミノ酸；並びに
（ｂ）２５６位にグルタミン酸（Ｅ）アミノ酸、若しくは２５６位にセリン（Ｓ）アミノ酸、若しくは２５６位にアルギニン（Ｒ）アミノ酸、若しくは２５６位にグルタミン（Ｑ）アミノ酸、若しくは２５６位にアスパラギン酸（Ｄ）アミノ酸
をさらに有し、
ここで、前記アミノ酸番号付けは、Ｋａｂａｔに記載のＥＵインデックスに従う、１〜４のいずれか１項に記載のポリペプチド。
[１０] （ａ）２５２位にチロシン（Ｙ）アミノ酸、及び２５４位にトレオニン（Ｔ）アミノ酸；又は、
（ｂ）２５４位にトレオニン（Ｔ）アミノ酸及び２５６位にグルタミン酸（Ｅ）アミノ酸；又は、
（ｃ）２５２位にチロシン（Ｙ）アミノ酸及び２５６位にグルタミン酸（Ｅ）アミノ酸をさらに有し、
ここで、前記アミノ酸番号付けは、Ｋａｂａｔに記載のＥＵインデックスに従う、１〜４のいずれか１項に記載のポリペプチド。
[１１] ２５２位にチロシン（Ｙ）アミノ酸、２５４位にトレオニン（Ｔ）アミノ酸、及び２５６位にグルタミン酸（Ｅ）アミノ酸をさらに有し、ここで、前記アミノ酸番号付けは、Ｋａｂａｔに記載のＥＵインデックスに従う、１〜４のいずれか１項に記載のポリペプチド。
[１２] （ａ）２３４位にフェニルアラニン（Ｆ）アミノ酸；
（ｂ）２３５位にグルタミン（Ｑ）アミノ酸；
（ｃ）３２２位にグルタミン（Ｑ）アミノ酸；
（ｄ）２５２位にチロシン（Ｙ）アミノ酸；
（ｅ）２５４位にトレオニン（Ｔ）アミノ酸；及び、
（ｆ）２５６位にグルタミン酸（Ｅ）アミノ酸
を有し、
ここで、前記アミノ酸番号付けは、Ｋａｂａｔに記載のＥＵインデックスに従う、１に記載のポリペプチド。
[１３] （ａ）２３４位にフェニルアラニン（Ｆ）アミノ酸；
（ｂ）２３５位にグルタミン（Ｑ）アミノ酸；
（ｃ）３３１位にグリシン（Ｑ）アミノ酸；
（ｄ）２５２位にチロシン（Ｙ）アミノ酸；
（ｅ）２５４位にトレオニン（Ｔ）アミノ酸；及び、
（ｆ）２５６位にグルタミン酸（Ｅ）アミノ酸
を有し、
ここで、前記アミノ酸番号付けは、Ｋａｂａｔに記載のＥＵインデックスに従う、１に記載のポリペプチド。
[１４] （ａ）２３４位にフェニルアラニン（Ｆ）アミノ酸；
（ｂ）２３５位にアラニン（Ａ）アミノ酸；
（ｃ）３２２位にグルタミン（Ｑ）アミノ酸；
（ｄ）２５２位にチロシン（Ｙ）アミノ酸；
（ｅ）２５４位にトレオニン（Ｔ）アミノ酸；及び、
（ｆ）２５６位にグルタミン酸（Ｅ）アミノ酸
を有し、
ここで、前記アミノ酸番号付けは、Ｋａｂａｔに記載のＥＵインデックスに従う、１に記載のポリペプチド。
[１５] 前記ポリペプチドが、野生型Ｆｃドメインを含む同じポリペプチドと比較して、向上した薬物動態（ＰＫ）特性を有する、１〜１４のいずれか１項に記載のポリペプチド。
[１６] 前記ＰＫ特性が、半減期である、１５に記載のポリペプチド。
[１７] 前記ポリペプチドが、野生型Ｆｃドメインを含む同じポリペプチドと比較して、向上したＦｃＲｎ結合を有する、１〜１６のいずれか１項に記載のポリペプチド。
[１８] 前記ＩｇＧ Ｆｃドメインが、非ヒト由来である、１〜１７のいずれか１項に記載のポリペプチド。
[１９] 前記ＩｇＧ Ｆｃドメインが、ヒト由来である、１〜１７のいずれか１項に記載のポリペプチド。
[２０] 前記非ヒトＩｇＧ Ｆｃドメインが、げっ歯類、ロバ、ヒツジ、ウサギ、ヤギ、モルモット、ラクダ、ウマ又はニワトリに由来する、１８に記載のポリペプチド。
[２１] 前記ＩｇＧ Ｆｃドメインが、ヒト免疫グロブリンＧクラス１（ＩｇＧ _１ ）Ｆｃドメイン、ヒト免疫グロブリンＧクラス２（ＩｇＧ _２ ）Ｆｃドメイン、ヒト免疫グロブリンＧクラス３（ＩｇＧ _３ ）Ｆｃドメイン、及びヒト免疫グロブリンＧクラス４（ＩｇＧ _４ ）Ｆｃドメインからなる群から選択される、１９に記載のポリペプチド。
[２２] 前記ポリペプチドが、さらに抗原結合ドメインを含む、１〜２１のいずれか１項に記載のポリペプチド。
[２３] 前記抗原結合ドメインが、モノクローナル抗体又はその抗原結合フラグメントに由来する、２２に記載のポリペプチド。
[２４] 前記抗原結合ドメインが、ヒト抗体、ヒト化抗体、又はキメラ抗体に由来する、２２に記載のポリペプチド。
[２５] 前記抗原結合ドメインが、以下：
（ａ）一本鎖抗体；
（ｂ）ダイアボディ；
（ｃ）抗体のポリペプチド鎖；
（ｄ）Ｆ（ａｂ’） _２ フラグメント；又は、
（ｅ）Ｆ（ａｂ）フラグメント
を含む、２２に記載のポリペプチド。
[２６] 前記ポリペプチドが、野生型Ｆｃドメインを含む同じポリペプチドと比較して、低減したＦｃ媒介エフェクター機能を有する、１〜２１のいずれか１項に記載のポリペプチド。
[２７] 前記エフェクター機能が、抗体依存性細胞媒介細胞傷害（ＡＤＣＣ）である、２６に記載のポリペプチド。
[２８] 前記エフェクター機能が、補体依存性細胞傷害（ＣＤＣ）である、２６に記載のポリペプチド。
[２９] 前記ポリペプチドが、野生型Ｆｃドメインを含む同じポリペプチドと比較して、Ｆｃγ受容体（ＦｃγＲ）に対し低い親和性を有する、１〜２８のいずれか１項に記載のポリペプチド。
[３０] 前記ＦｃγＲが、ヒトＦｃγＲである、２９に記載のポリペプチド。
[３１] 前記ＦｃγＲが、ＦｃγＲＩ、ＦｃγＲＩＩ、及びＦｃγＲＩＩＩからなる群から選択される、２９に記載のポリペプチド。
[３２] 前記ＦｃγＲＩが、ＦｃγＲＩαである、３１に記載のポリペプチド。
[３３] 前記ＦｃγＲＩＩが、ＦｃγＲＩＩａ又はＦｃγＲＩＩｂである、３１に記載のポリペプチド。
[３４] 前記ＦｃγＲＩＩＩが、ＦｃγＲＩＩＩ（１５８Ｖ）又はＦｃγＲＩＩＩ（１５８Ｆ）である、３１に記載のポリペプチド。
[３５] 前記ポリペプチドが、野生型Ｆｃドメインを含む同じポリペプチドと比較して、向上した親和性でＦｃＲｎと結合する、１〜３４のいずれか１項に記載のポリペプチド。
[３６] 前記ポリペプチドが、ｐＨ７．４におけるよりもｐＨ６．０において、ＦｃＲｎに対する高い親和性を有する、３５に記載のポリペプチド。
[３７] 前記ポリペプチドが、野生型Ｆｃドメインを含む同じポリペプチドと比較して、低減した親和性でＣ１ｑと結合する、１〜３６のいずれか１項に記載のポリペプチド。
[３８] 前記ポリペプチドが、ＦＥＳ−ＹＴＥ ＩｇＧ Ｆｃドメインを含む同じポリペプチドと比較して、熱安定性の増加を呈示する、１〜３７のいずれか１項に記載のポリペプチド。
[３９] 前記熱安定性が、示差走査熱量測定（ＤＳＣ）により測定される、３８に記載のポリペプチド。
[４０] 前記熱安定性の増加が、少なくとも４℃である、３９に記載のポリペプチド。
[４１] 前記熱安定性が、示差走査型蛍光定量法（ＤＳＦ）により測定される、３８に記載のポリペプチド。
[４２] 前記ＤＳＦ蛍光プローブが、Ｓｙｐｒｏ Ｏｒａｎｇｅである、４１に記載のポリペプチド。
[４３] 前記熱安定性の増加が、少なくとも５℃である、４２に記載のポリペプチド。
[４４] 前記ポリペプチドが、ＦＥＳ−ＹＴＥ ＩｇＧ Ｆｃドメインを含む同じポリペプチドと比較して、ポリエチレングリコール（ＰＥＧ）沈殿アッセイを用いて測定される見かけ溶解度の増加を呈示する、１〜４３のいずれか１項に記載のポリペプチド。
[４５] 前記ポリペプチドが、ＦＥＳ−ＹＴＥ ＩｇＧ Ｆｃドメインを含む同じポリペプチドと比較して、加速安定性アッセイを用いて測定される安定性の増加を呈示する、１〜４４のいずれか１項に記載のポリペプチド。
[４６] 前記加速安定性アッセイが、（ｉ）長期間にわたるポリペプチドのインキュベーション、及び（ｉｉ）高温でのインキュベーションを含む、４５に記載のポリペプチド。
[４７] 前記加速安定性アッセイが、高濃度でのインキュベーションにより実施される、４６に記載のポリペプチド。
[４８] 前記長期間が、少なくとも１ヵ月である、４６に記載のポリペプチド。
[４９] 前記高濃度が、少なくとも２５ｍｇ／ｍｌである、４７に記載のポリペプチド。
[５０] 前記高温が、少なくとも４０℃である、４６に記載のポリペプチド。
[５１] 前記加速安定性アッセイが、高機能サイズ排除クロマトグラフィー（ＨＰＳＥＣ）又は動的光散乱法（ＤＬＳ）を用いて実施される、４５〜５０のいずれか１項に記載のポリペプチド。
[５２] １〜５１のいずれか１項に記載のポリペプチドをコードする配列を含む、単離された核酸。
[５３] ５２に記載の核酸を含む組成物。
[５４] ５２に記載の核酸を含む発現ベクター。
[５５] ５２に記載の核酸、５３に記載の組成物、又は５４に記載のベクターを含む宿主細胞。
[５６] （ａ）５５に記載の細胞を培養するステップ；及び（ｂ）前記ポリペプチドを単離するステップを含む、１〜５１のいずれか１項に記載のポリペプチドを作製する方法。
[５７] １〜５１のいずれか１項に記載のポリペプチドと、担体とを含む組成物。
[５８] １〜５１のいずれか１項に記載のポリペプチドを含む診断薬。
[５９] 前記ポリペプチドが標識されている、５８に記載の診断薬。
[６０] １〜５１のいずれか１項に記載のポリペプチドと、治療部分とを含むコンジュゲート。
[６１] １〜５１のいずれか１項に記載のポリペプチド、又は５２〜５５若しくは５７〜６０のいずれか１項に記載の組成物を含むキット。
[６２] 哺乳動物を治療する方法であって、１〜５１のいずれか１項に記載のポリペプチド、又は５２〜５５若しくは５７〜６０のいずれか１項に記載の組成物を有効量で、治療が必要な哺乳動物に投与することを含む方法。
[６３] Ｆｃドメインを含む親ポリペプチドのＦｃ誘導性エフェクター機能を低減する方法であって、以下：
（ａ）Ｆｃドメインにおける２３４位の前記アミノ酸をフェニルアラニン（Ｆ）で置換するステップ；
（ｂ）Ｆｃドメインにおける２３５位の前記アミノ酸をアラニン（Ａ）、アスパラギン（Ｎ）、フェニルアラニン（Ｆ）、グルタミン（Ｑ）、若しくはバリン（Ｖ）で置換するステップ；及び
（ｃ）Ｆｃドメインの３２２位の前記アミノ酸をアラニン（Ａ）、アスパラギン酸（Ｄ）、グルタミン酸（Ｅ）、ヒスチジン（Ｈ）、アスパラギン（Ｎ）、若しくはグルタミン（Ｑ）で置換するステップ；又はＦｃドメインの３３１位のアミノ酸をアラニン（Ａ）若しくはグリシン（Ｇ）で置換するステップ
を含み、
ここで、前記Ｆｃドメインの前記アミノ酸番号付けは、Ｋａｂａｔに記載のＥＵインデックスに従う方法。
[６４] 前記親ポリペプチドの前記Ｆｃドメインが、
（ａ）２５２位にチロシン（Ｙ）アミノ酸、若しくは２５２位にセリン（Ｓ）アミノ酸、若しくは２５２位にトリプトファン（Ｗ）アミノ酸、若しくは２５２位にトレオニン（Ｔ）アミノ酸；及び／又は
（ｂ）２５４位にトレオニン（Ｔ）アミノ酸；及び／又は
（ｃ）２５６位にグルタミン酸（Ｅ）アミノ酸、若しくは２５６位にセリン（Ｓ）アミノ酸、若しくは２５６位にアルギニン（Ｒ）アミノ酸、若しくは２５６位にグルタミン（Ｑ）アミノ酸、若しくは２５６位にアスパラギン酸（Ｄ）アミノ酸
を有し、
ここで、前記Ｆｃドメインの前記アミノ酸番号付けは、Ｋａｂａｔに記載のＥＵインデックスに従う方法。
[６５] 前記親ポリペプチドの前記Ｆｃドメインが、
（ａ）２５２位にチロシン（Ｙ）；及び／又は
（ｂ）２５４位にトレオニン（Ｔ）；及び／又は
（ｃ）２５６位にグルタミン酸（Ｅ）
を有し、
ここで、前記Ｆｃドメインの前記アミノ酸番号付けは、Ｋａｂａｔに記載のＥＵインデックスに従う方法。
[６６] Ｆｃドメインを含む親ポリペプチドのＦｃ誘導性エフェクター機能を低減すると共に、その半減期を増加させる方法であって、以下：
（ａ）前記Ｆｃドメインにおける２３４位の前記アミノ酸をフェニルアラニン（Ｆ）で置換するステップ；
（ｂ）前記Ｆｃドメインにおける２３５位の前記アミノ酸をアラニン（Ａ）、アスパラギン（Ｎ）、フェニルアラニン（Ｆ）、グルタミン（Ｑ）、若しくはバリン（Ｖ）で置換するステップ；及び
（ｃ）前記Ｆｃドメインの３２２位の前記アミノ酸をアラニン（Ａ）、アスパラギン酸（Ｄ）、グルタミン酸（Ｅ）、ヒスチジン（Ｈ）、アスパラギン（Ｎ）、若しくはグルタミン（Ｑ）で置換するステップ；又は前記Ｆｃドメインの３３１位のアミノ酸をアラニン（Ａ）若しくはグリシン（Ｇ）で置換するステップ；並びに
（ｄ）２５２位の前記アミノ酸をチロシン（Ｙ）若しくはセリン（Ｓ）若しくはトリプトファン（Ｗ）若しくはトレオニン（Ｔ）で置換するステップ
を含み；
ここで、前記Ｆｃドメインの前記アミノ酸番号付けは、Ｋａｂａｔに記載のＥＵインデックスに従う方法。
[６７] ２５２位の前記アミノ酸がチロシン（Ｙ）で置換され、ここで、前記アミノ酸番号付けは、Ｋａｂａｔに記載のＥＵインデックスに従う、６６に記載の方法。
[６８] （ａ）２５４位の前記アミノ酸をトレオニン（Ｔ）で置換するステップ；及び、
（ｂ）２５６位の前記アミノ酸をグルタミン酸（Ｅ）若しくはセリン（Ｓ）、若しくはアルギニン（Ｒ）、若しくはグルタミン（Ｑ）で置換するステップ
をさらに含み、
ここで、前記Ｆｃドメインの前記アミノ酸番号付けは、Ｋａｂａｔに記載のＥＵインデックスに従う、６６又は６７に記載の方法。
[６９] ２５６位の前記アミノ酸がグルタミン酸（Ｅ）で置換され、ここで、前記アミノ酸番号付けは、Ｋａｂａｔに記載のＥＵインデックスに従う、６８に記載の方法。
[７０] ２３４位の前記アミノ酸がフェニルアラニン（Ｆ）で置換され；２３５位の前記アミノ酸がグルタミン（Ｑ）で置換され；３２２位の前記アミノ酸がグルタミン（Ｑ）で置換され、ここで、前記アミノ酸番号付けは、Ｋａｂａｔに記載のＥＵインデックスに従う、６３〜６９のいずれか１項に記載の方法。
[７１] ２３４位の前記アミノ酸がフェニルアラニン（Ｆ）で置換され；２３５位の前記アミノ酸がグルタミン（Ｑ）で置換され；３３１位の前記アミノ酸がグリシン（Ｇ）で置換され、ここで、前記アミノ酸番号付けは、Ｋａｂａｔに記載のＥＵインデックスに従う、６３〜７０のいずれか１項に記載の方法。
[７２] ２３４位の前記アミノ酸がフェニルアラニン（Ｆ）で置換され；２３５位の前記アミノ酸がアラニン（Ａ）で置換され；３２２位の前記アミノ酸がグルタミン（Ｑ）で置換され、ここで、前記アミノ酸番号付けは、Ｋａｂａｔに記載のＥＵインデックスに従う、６３〜７０のいずれか１項に記載の方法。
[７３] 前記エフェクター機能が、抗体依存性細胞媒介細胞傷害（ＡＤＣＣ）である、６３〜７２のいずれか１項に記載の方法。
[７４] 前記エフェクター機能が、補体依存性細胞傷害（ＣＤＣ）である、６３〜７２のいずれか１項に記載の方法。
[７５] 前記ポリペプチドが、ＦＥＳ−ＹＴＥ ＩｇＧ Ｆｃドメインを含む同じポリペプチドと比較して、熱安定性の増加を呈示する、６３〜７２のいずれか１項に記載の方法。
[７６] 前記熱安定性が、示差走査熱量測定（ＤＳＣ）により測定される、７５に記載の方法。
[７７] 前記熱安定性の増加が、少なくとも４℃である、７５に記載の方法。
[７８] 前記熱安定性が、ＤＳＦ蛍光プローブを用いた示差走査蛍光定量法（ＤＳＦ）により測定される、７５に記載の方法。
[７９] 前記ＤＳＦ蛍光プローブが、Ｓｙｐｒｏ Ｏｒａｎｇｅである、７８に記載の方法。
[８０] 前記熱安定性の増加が、少なくとも５℃である、７８に記載の方法。
[８１] 前記ポリペプチドが、ＦＥＳ−ＹＴＥ ＩｇＧ Ｆｃドメインを含む同じポリペプチドと比較して、ポリエチレングリコール（ＰＥＧ）沈殿アッセイを用いて測定される見かけ溶解度の増加を呈示する、６３〜７２のいずれか１項に記載の方法。
[８２] 前記ポリペプチドが、ＦＥＳ−ＹＴＥ ＩｇＧ Ｆｃドメインを含む同じポリペプチドと比較して、加速安定性アッセイを用いて測定される安定性の増加を呈示する、６３〜７２のいずれか１項に記載の方法。
[８３] 前記加速安定性アッセイが、（ｉ）長期間にわたるポリペプチドのインキュベーション、及び（ｉｉ）高温でのインキュベーションを含む、８２に記載の方法。
[８４] 前記加速安定性アッセイが、高濃度でのインキュベーションにより実施される、８３に記載の方法。
[８５] 前記長期間が、少なくとも１ヵ月である、８３に記載の方法。
[８６] 前記高濃度が、少なくとも２５ｍｇ／ｍｌである、８４に記載の方法。
[８７] 前記高温が、少なくとも４０℃である、８３に記載の方法。 All publications, patents, patent applications, or other references cited in this application may be incorporated by reference for any purpose, as if each individual publication, patent, patent application, or other reference was included. They are incorporated herein by reference in their entirety for all purposes, to the same extent as indicated individually. In addition, US Provisional Patent Application No. 61 / 640,327, filed Apr. 30, 2012, is hereby incorporated by reference in its entirety for all purposes.

[1] An isolated polypeptide comprising a mutant IgG Fc domain, wherein the mutant IgG Fc domain is
(A) a phenylalanine (F) amino acid at position 234;
(B) an alanine (A), asparagine (N), phenylalanine (F), glutamine (Q), or valine (V) amino acid at position 235; and
(C) alanine (A), aspartic acid (D), glutamic acid (E), histidine (H), asparagine (N), or glutamine (Q) amino acid at position 322; or alanine (A) or glycine at position 331 ( G) Amino acids
Have
Wherein the amino acid numbering is an isolated polypeptide according to the EU index described in Kabat.
[2] Has a phenylalanine (F) amino acid at position 234; a glutamine (Q) amino acid at position 235; and a glutamine (Q) amino acid at position 322, where the amino acid numbering is according to the EU index described in Kabat 1. The polypeptide according to 1.
[3] It has a phenylalanine (F) amino acid at position 234; a glutamine (Q) amino acid at position 235; and a glycine (G) amino acid at position 331, where the amino acid numbering is according to the EU index described in Kabat 1. The polypeptide according to 1.
[4] Has a phenylalanine (F) amino acid at position 234; an alanine (A) amino acid at position 235; and a glutamine (Q) amino acid at position 322, where the amino acid numbering is according to the EU index set forth in Kabat 1. The polypeptide according to 1.
[5] (a) tyrosine (Y) amino acid at position 252; serine (S) amino acid at position 252; tryptophan (W) amino acid at position 252; or threonine (T) amino acid at position 252; and / or
(B) a threonine (T) amino acid at position 254; and / or
(C) glutamic acid (E) amino acid at position 256, serine (S) amino acid at position 256, arginine (R) amino acid at position 256, glutamine (Q) amino acid at position 256, or aspartic acid at position 256 (D )amino acid
Further comprising
Here, the amino acid numbering is the polypeptide according to any one of 1 to 4, according to the EU index described in Kabat.
[6] (a) a tyrosine (Y) amino acid at position 252; and / or
(B) a threonine (T) amino acid at position 254; and / or
(C) Glutamic acid (E) amino acid at position 256
Further comprising
Here, the amino acid numbering is the polypeptide according to any one of 1 to 4, according to the EU index described in Kabat.
[7] (a) tyrosine (Y) amino acid at position 252; serine (S) amino acid at position 252; tryptophan (W) amino acid at position 252; or threonine (T) amino acid at position 252;
(B) Threonine (T) amino acid at position 254
Further comprising
Here, the amino acid numbering is the polypeptide according to any one of 1 to 4, according to the EU index described in Kabat.
[8] (a) a threonine (T) amino acid at position 254; and
(B) glutamic acid (E) amino acid at position 256, serine (S) amino acid at position 256, arginine (R) amino acid at position 256, glutamine (Q) amino acid at position 256, or aspartic acid at position 256 (D )amino acid
Further comprising
Here, the amino acid numbering is the polypeptide according to any one of 1 to 4, according to the EU index described in Kabat.
[9] (a) tyrosine (Y) amino acid at position 252; serine (S) amino acid at position 252; tryptophan (W) amino acid at position 252; or threonine (T) amino acid at position 252;
(B) glutamic acid (E) amino acid at position 256, serine (S) amino acid at position 256, arginine (R) amino acid at position 256, glutamine (Q) amino acid at position 256, or aspartic acid at position 256 (D )amino acid
Further comprising
Here, the amino acid numbering is the polypeptide according to any one of 1 to 4, according to the EU index described in Kabat.
[10] (a) a tyrosine (Y) amino acid at position 252 and a threonine (T) amino acid at position 254; or
(B) a threonine (T) amino acid at position 254 and a glutamic acid (E) amino acid at position 256; or
(C) further having a tyrosine (Y) amino acid at position 252 and a glutamic acid (E) amino acid at position 256;
Here, the amino acid numbering is the polypeptide according to any one of 1 to 4, according to the EU index described in Kabat.
[11] A tyrosine (Y) amino acid at position 252; a threonine (T) amino acid at position 254; and a glutamic acid (E) amino acid at position 256, wherein the amino acid numbering is an EU index described in Kabat 5. The polypeptide according to any one of 1 to 4, according to
[12] (a) a phenylalanine (F) amino acid at position 234;
(B) a glutamine (Q) amino acid at position 235;
(C) a glutamine (Q) amino acid at position 322;
(D) a tyrosine (Y) amino acid at position 252;
(E) a threonine (T) amino acid at position 254; and
(F) Glutamic acid (E) amino acid at position 256
Have
The polypeptide according to 1, wherein the amino acid numbering is according to the EU index described in Kabat.
[13] (a) a phenylalanine (F) amino acid at position 234;
(B) a glutamine (Q) amino acid at position 235;
(C) a glycine (Q) amino acid at position 331;
(D) a tyrosine (Y) amino acid at position 252;
(E) a threonine (T) amino acid at position 254; and
(F) Glutamic acid (E) amino acid at position 256
Have
The polypeptide according to 1, wherein the amino acid numbering is according to the EU index described in Kabat.
[14] (a) a phenylalanine (F) amino acid at position 234;
(B) an alanine (A) amino acid at position 235;
(C) a glutamine (Q) amino acid at position 322;
(D) a tyrosine (Y) amino acid at position 252;
(E) a threonine (T) amino acid at position 254; and
(F) Glutamic acid (E) amino acid at position 256
Have
The polypeptide according to 1, wherein the amino acid numbering is according to the EU index described in Kabat.
[15] The polypeptide of any one of 1-14, wherein the polypeptide has improved pharmacokinetic (PK) properties compared to the same polypeptide comprising a wild-type Fc domain.
[16] The polypeptide according to 15, wherein the PK characteristic is a half-life.
[17] The polypeptide of any one of 1-16, wherein the polypeptide has improved FcRn binding compared to the same polypeptide comprising a wild-type Fc domain.
[18] The polypeptide according to any one of 1 to 17, wherein the IgG Fc domain is derived from non-human.
[19] The polypeptide according to any one of 1 to 17, wherein the IgG Fc domain is derived from a human.
[20] The polypeptide according to 18, wherein the non-human IgG Fc domain is derived from a rodent, a donkey, a sheep, a rabbit, a goat, a guinea pig, a camel, a horse, or a chicken.
[21] The IgG Fc domain is a human immunoglobulin G class 1 (IgG ₁ ) Fc domain, a human immunoglobulin G class 2 (IgG ₂ ) Fc domain, a human immunoglobulin G class 3 (IgG ₃ ) Fc domain, and a human 20. The polypeptide according to 19, which is selected from the group consisting of an immunoglobulin G class 4 (IgG ₄ ) Fc domain.
[22] The polypeptide according to any one of 1 to 21, wherein the polypeptide further comprises an antigen-binding domain.
[23] The polypeptide according to 22, wherein the antigen-binding domain is derived from a monoclonal antibody or an antigen-binding fragment thereof.
[24] The polypeptide according to 22, wherein the antigen-binding domain is derived from a human antibody, a humanized antibody, or a chimeric antibody.
[25] The antigen-binding domain is:
(A) a single chain antibody;
(B) Diabody;
(C) an antibody polypeptide chain;
(D) F (ab ′) ₂ fragment; or
(E) F (ab) fragment
The polypeptide according to 22, comprising
[26] The polypeptide of any one of 1-21, wherein the polypeptide has a reduced Fc-mediated effector function compared to the same polypeptide comprising a wild-type Fc domain.
[27] The polypeptide according to 26, wherein the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC).
[28] The polypeptide according to 26, wherein the effector function is complement-dependent cytotoxicity (CDC).
[29] The polypeptide according to any one of 1 to 28, wherein the polypeptide has a low affinity for an Fcγ receptor (FcγR) compared to the same polypeptide comprising a wild-type Fc domain.
[30] The polypeptide according to 29, wherein the FcγR is human FcγR.
[31] The polypeptide according to 29, wherein the FcγR is selected from the group consisting of FcγRI, FcγRII, and FcγRIII.
[32] The polypeptide according to 31, wherein the FcγRI is FcγRIα.
[33] The polypeptide according to 31, wherein the FcγRII is FcγRIIa or FcγRIIb.
[34] The polypeptide according to 31, wherein the FcγRIII is FcγRIII (158V) or FcγRIII (158F).
[35] The polypeptide of any one of 1-34, wherein the polypeptide binds FcRn with improved affinity compared to the same polypeptide comprising a wild-type Fc domain.
[36] The polypeptide according to 35, wherein the polypeptide has a higher affinity for FcRn at pH 6.0 than at pH 7.4.
[37] The polypeptide of any one of 1-36, wherein the polypeptide binds C1q with reduced affinity compared to the same polypeptide comprising a wild-type Fc domain.
[38] The polypeptide of any one of 1-37, wherein the polypeptide exhibits increased thermal stability compared to the same polypeptide comprising a FES-YTE IgG Fc domain.
[39] The polypeptide according to 38, wherein the thermal stability is measured by differential scanning calorimetry (DSC).
[40] The polypeptide according to 39, wherein the increase in thermal stability is at least 4 ° C.
[41] The polypeptide according to 38, wherein the thermal stability is measured by differential scanning fluorimetry (DSF).
[42] The polypeptide according to 41, wherein the DSF fluorescent probe is Sypro Orange.
[43] The polypeptide according to 42, wherein the increase in thermal stability is at least 5 ° C.
[44] Any of 1-43, wherein said polypeptide exhibits an increase in apparent solubility as measured using a polyethylene glycol (PEG) precipitation assay compared to the same polypeptide comprising a FES-YTE IgG Fc domain The polypeptide according to claim 1.
[45] Any one of 1-44, wherein the polypeptide exhibits an increase in stability as measured using an accelerated stability assay compared to the same polypeptide comprising a FES-YTE IgG Fc domain. The polypeptide according to 1.
[46] The polypeptide of 45, wherein the accelerated stability assay comprises (i) incubation of the polypeptide for an extended period of time, and (ii) incubation at an elevated temperature.
[47] The polypeptide of 46, wherein the accelerated stability assay is performed by incubation at a high concentration.
[48] The polypeptide according to 46, wherein the long period is at least one month.
[49] The polypeptide according to 47, wherein the high concentration is at least 25 mg / ml.
[50] The polypeptide according to 46, wherein the high temperature is at least 40 ° C.
[51] The polypeptide according to any one of 45 to 50, wherein the accelerated stability assay is performed using high-performance size exclusion chromatography (HPSEC) or dynamic light scattering (DLS).
[52] An isolated nucleic acid comprising a sequence encoding the polypeptide according to any one of 1 to 51.
[53] A composition comprising the nucleic acid according to 52.
[54] An expression vector comprising the nucleic acid according to 52.
[55] A host cell comprising the nucleic acid according to 52, the composition according to 53, or the vector according to 54.
[56] A method for producing the polypeptide according to any one of 1 to 51, comprising (a) culturing the cell according to 55; and (b) isolating the polypeptide.
[57] A composition comprising the polypeptide according to any one of 1 to 51 and a carrier.
[58] A diagnostic agent comprising the polypeptide according to any one of 1 to 51.
[59] The diagnostic agent according to 58, wherein the polypeptide is labeled.
[60] A conjugate comprising the polypeptide of any one of 1 to 51 and a therapeutic moiety.
[61] A kit comprising the polypeptide according to any one of 1 to 51, or the composition according to any one of 52 to 55 or 57 to 60.
[62] A method for treating a mammal, comprising an effective amount of the polypeptide according to any one of 1 to 51, or the composition according to any one of 52 to 55 or 57 to 60, Administering to a mammal in need of treatment.
[63] A method of reducing Fc-induced effector function of a parent polypeptide comprising an Fc domain, comprising:
(A) replacing the amino acid at position 234 in the Fc domain with phenylalanine (F);
(B) replacing the amino acid at position 235 in the Fc domain with alanine (A), asparagine (N), phenylalanine (F), glutamine (Q), or valine (V);
(C) replacing the amino acid at position 322 of the Fc domain with alanine (A), aspartic acid (D), glutamic acid (E), histidine (H), asparagine (N), or glutamine (Q); or Fc Substituting the amino acid at position 331 of the domain with alanine (A) or glycine (G)
Including
Here, the amino acid numbering of the Fc domain is a method according to the EU index described in Kabat.
[64] the Fc domain of the parent polypeptide is
(A) a tyrosine (Y) amino acid at position 252; a serine (S) amino acid at position 252; a tryptophan (W) amino acid at position 252; or a threonine (T) amino acid at position 252; and / or
(B) a threonine (T) amino acid at position 254; and / or
(C) glutamic acid (E) amino acid at position 256, serine (S) amino acid at position 256, arginine (R) amino acid at position 256, glutamine (Q) amino acid at position 256, or aspartic acid at position 256 (D )amino acid
Have
Here, the amino acid numbering of the Fc domain is a method according to the EU index described in Kabat.
[65] the Fc domain of the parent polypeptide is
(A) tyrosine (Y) at position 252; and / or
(B) threonine (T) at position 254; and / or
(C) Glutamic acid at position 256 (E)
Have
Here, the amino acid numbering of the Fc domain is a method according to the EU index described in Kabat.
[66] A method of reducing the Fc-induced effector function of a parent polypeptide comprising an Fc domain and increasing its half-life, comprising:
(A) substituting the amino acid at position 234 in the Fc domain with phenylalanine (F);
(B) replacing the amino acid at position 235 in the Fc domain with alanine (A), asparagine (N), phenylalanine (F), glutamine (Q), or valine (V);
(C) substituting the amino acid at position 322 of the Fc domain with alanine (A), aspartic acid (D), glutamic acid (E), histidine (H), asparagine (N), or glutamine (Q); or Substituting the amino acid at position 331 of the Fc domain with alanine (A) or glycine (G); and
(D) replacing the amino acid at position 252 with tyrosine (Y), serine (S), tryptophan (W), or threonine (T);
Including:
Here, the amino acid numbering of the Fc domain is a method according to the EU index described in Kabat.
[67] The method of 66, wherein the amino acid at position 252 is substituted with tyrosine (Y), wherein the amino acid numbering is according to the EU index set forth in Kabat.
[68] (a) substituting the amino acid at position 254 with threonine (T); and
(B) substituting the amino acid at position 256 with glutamic acid (E), serine (S), arginine (R), or glutamine (Q)
Further including
68. The method according to 66 or 67, wherein the amino acid numbering of the Fc domain is according to the EU index described in Kabat.
[69] The method of 68, wherein the amino acid at position 256 is substituted with glutamic acid (E), wherein the amino acid numbering is according to the EU index set forth in Kabat.
[70] The amino acid at position 234 is replaced with phenylalanine (F); the amino acid at position 235 is replaced with glutamine (Q); the amino acid at position 322 is replaced with glutamine (Q), wherein the amino acid 70. The method according to any one of 63 to 69, wherein the numbering is in accordance with an EU index described in Kabat.
[71] The amino acid at position 234 is substituted with phenylalanine (F); the amino acid at position 235 is replaced with glutamine (Q); the amino acid at position 331 is replaced with glycine (G), wherein the amino acid 71. The method according to any one of 63 to 70, wherein the numbering is in accordance with an EU index described in Kabat.
[72] The amino acid at position 234 is substituted with phenylalanine (F); the amino acid at position 235 is replaced with alanine (A); the amino acid at position 322 is replaced with glutamine (Q), wherein the amino acid 71. The method according to any one of 63 to 70, wherein the numbering is in accordance with an EU index described in Kabat.
[73] The method of any one of 63 to 72, wherein the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC).
[74] The method according to any one of 63 to 72, wherein the effector function is complement-dependent cytotoxicity (CDC).
[75] The method of any one of 63-72, wherein the polypeptide exhibits increased thermal stability compared to the same polypeptide comprising a FES-YTE IgG Fc domain.
[76] The method of 75, wherein the thermal stability is measured by differential scanning calorimetry (DSC).
[77] The method of 75, wherein the increase in thermal stability is at least 4 ° C.
[78] The method according to 75, wherein the thermal stability is measured by a differential scanning fluorometry (DSF) using a DSF fluorescent probe.
[79] The method according to 78, wherein the DSF fluorescent probe is Sypro Orange.
[80] The method of 78, wherein the increase in thermal stability is at least 5 ° C.
[81] Any of 63-72, wherein said polypeptide exhibits an increase in apparent solubility as measured using a polyethylene glycol (PEG) precipitation assay compared to the same polypeptide comprising a FES-YTE IgG Fc domain The method according to claim 1.
[82] Any of 63-72, wherein said polypeptide exhibits an increase in stability as measured using an accelerated stability assay compared to the same polypeptide comprising a FES-YTE IgG Fc domain. The method described in 1.
[83] The method of 82, wherein the accelerated stability assay comprises (i) incubation of the polypeptide for an extended period of time, and (ii) incubation at an elevated temperature.
[84] The method of 83, wherein the accelerated stability assay is performed by incubation at a high concentration.
[85] The method of 83, wherein the long period is at least one month.
[86] The method of 84, wherein the high concentration is at least 25 mg / ml.
[87] A method according to 83, wherein the high temperature is at least 40 ° C.

Claims (12)

An isolated polypeptide comprising a variant IgG Fc domain, wherein the variant IgG Fc domain is
(A) a phenylalanine (F) amino acid at position 234;
(B) a glutamine (Q) amino acid at position 235;
(C) a glutamine (Q) amino acid at position 322;
(D) a tyrosine (Y) amino acid at position 252;
(E) a threonine (T) amino acid at position 254; and
(F) having a glutamic acid (E) amino acid at position 256;
Wherein the amino acid numbering is an isolated polypeptide according to the EU index described in Kabat. An isolated polypeptide comprising a variant IgG Fc domain, wherein the variant IgG Fc domain is
(A) a phenylalanine (F) amino acid at position 234;
(B) a glutamine (Q) amino acid at position 235;
(C) a glycine ( G ) amino acid at position 331;
(D) a tyrosine (Y) amino acid at position 252;
(E) a threonine (T) amino acid at position 254; and
(F) having a glutamic acid (E) amino acid at position 256;
Wherein the amino acid numbering is an isolated polypeptide according to the EU index described in Kabat. An isolated polypeptide comprising a variant IgG Fc domain, wherein the variant IgG Fc domain is
(A) a phenylalanine (F) amino acid at position 234;
(B) an alanine (A) amino acid at position 235;
(C) a glutamine (Q) amino acid at position 322;
(D) a tyrosine (Y) amino acid at position 252;
(E) a threonine (T) amino acid at position 254; and
(F) having a glutamic acid (E) amino acid at position 256;
Wherein the amino acid numbering is an isolated polypeptide according to the EU index described in Kabat. 4. The polypeptide of any one of claims 1-3 , wherein the polypeptide has an improved half-life compared to the same polypeptide comprising a wild type Fc domain. 5. The polypeptide of any one of claims 1-4 , wherein the polypeptide has improved FcRn binding compared to the same polypeptide comprising a wild type Fc domain. The polypeptide according to any one of claims 1 to 5 , wherein the polypeptide further comprises an antigen-binding domain or an antigen-binding domain derived from a monoclonal antibody or an antigen-binding fragment thereof. 7. The polypeptide of any one of claims 1-6 , wherein the polypeptide has reduced Fc-mediated effector function compared to the same polypeptide comprising a wild type Fc domain. 8. The polypeptide of any one of claims 1-7 , wherein the polypeptide has a lower affinity for an Fcγ receptor (FcγR) compared to the same polypeptide comprising a wild type Fc domain. 9. The polypeptide of claim 8 , wherein the polypeptide has a higher affinity for FcRn at pH 6.0 than at pH 7.4. A conjugate comprising the polypeptide of any one of claims 1 to 9 and a therapeutic moiety. Use of the polypeptide according to any one of claims 1 to 9 in the manufacture of a medicament for treating a mammal. A pharmaceutical composition for treating a mammal, comprising the polypeptide according to any one of claims 1 to 9 .

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